Sandra Radić

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.

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.

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.

Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.)

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1mM ammonium (NH(4)(+)) as the sole source of nitrogen. Growth of NH(4)(+)-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH(4)(+) medium with 25mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH(4)(+) induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH(4)(+) as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20mM) or Gln (10mM) in combination with NH(4)(+) (1mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin.

The efficiency of combined CaO/electrochemical treatment in removal of acid mine drainage induced toxicity and genotoxicity.

Acid mine drainage (AMD) is a by-product of the mining industry that has a detrimental effect on aquatic plant and animal life due to high load of heavy metals and sulfates. In the present study, the toxic and genotoxic potential of AMD prior to and following combination of neutralization/electrocoagulation processes was evaluated using several bioassays and selected parameters. Regardless of pH correction of AMD prior to Daphnia bioassay, high acute toxicity was observed in Daphnia magna. The mine leachate also induced strong phyto-, cyto- and genotoxicity to Allium cepa roots. Short term exposure to AMD inhibited duckweed growth and chlorophyll a content and simultaneously promoted lipid peroxidation and DNA damage despite duckweed capability to upregulate antioxidative defense mechanisms. The results show that observed (geno)toxicity could be related to oxidative stress most probably induced by toxic metal action. However, influence of low pH as a contributing factor in the phytotoxicity of AMD cannot be excluded. The application of combined treatment eliminated genotoxicity and was highly efficient in reducing toxicity of AMD. Thus, the method seems to be suitable for treatment of AMD waters enabling their safe discharge to an aquatic environment.

Assessment of surface water in the vicinity of fertilizer factory using fish and plants

The genotoxic and toxic potential of polluted surface water exposed to a fertilizer factory effluent was evaluated using assays with fish (Cyprinus carpio) and plant (Lemna minor) model organisms. Beside classical physicochemical parameters, the contents of fluorides, some heavy metals and polycyclic aromatic hydrocarbons were analyzed as well. Surface water caused inhibition of plant growth and decrease of photosynthetic pigment content. Regarding DNA damage and oxidative stress parameters, both fish and plants showed similar response to the surface water. In confirmation to biochemical markers, histopathological analysis of gill and liver tissues revealed a higher incidence of lesions in fish exposed to polluted surface water. Generally, results obtained by biological monitoring were mostly in agreement with chemical analysis of the surface water, although several discrepancies were observed which might be due to difference in sensitivity of model organisms or in experimental conditions (laboratory and field exposure). The results imply that conventional chemical analysis should be extended to genotoxicity/toxicity assays as measured biological effects and the potential health hazard cannot be predicted based on the physicochemical characteristics of water samples alone.

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.

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.