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With the aim to characterize changes caused by grapevine leafroll-associated virus 3 (GLRaV-3) singly or in coinfection with other viruses and to potentially determine genotype-specific or common markers of viral infection, thirty-six parameters, including nutrient status, oxidative stress parameters, and primary metabolism as well as symptoms incidence were investigated in ‘Cabernet Franc,’ ‘Merlot,’ ‘Pinot Noir,’ and ‘Tribidrag’ grapevine varieties. Host responses were characterized by changes in cellular redox state rather than disturbances in nutrient status and primary metabolic processes. Superoxide dismutase, hydrogen peroxide, and proteins were drastically affected regardless of the type of isolate, the host, and the duration of the infection, so they present cellular markers of viral infection. No clear biological pattern could be ascertained for each of the GLRaV-3 genotypes. There is a need to provide a greater understanding of virus epidemiology in viticulture due to the increasing natural disasters and climate change to provide for global food production security. Finding grape varieties that will be able to cope with those changes can aid in this task. Among the studied grapevine varieties, autochthonous ‘Tribidrag’ seems to be more tolerant to symptoms development despite numerous physiological changes caused by viruses.

Coal ash deposited in open landfills is a potential source of environmental pollutants due to the contained toxic element content. The weathered coal ash used in this study additionally contains enhanced activity concentrations of 238U series radionuclides. This study aimed to determine the physiological effects of enhanced ionizing radiation and toxic elements on five plant species (smilo grass, sticky fleabane, blackberry, mastic and pine tree) inhabiting the coal ash disposal site. Among the potentially toxic measured elements, contents of Sb, As and especially V significantly exceeded their respective levels at the control site, as well as the content of 238U and its progenies. Significant changes in photosynthetic pigments were recorded following chronic exposure to the plants growing on the coal ash site. Different responses were also observed in the plant species regarding the activity of catalase and glutathione-S-transferase (GST). The level of lipid peroxidation markedly increased in plants from the disposal site, except in blackberry, wherein GST activity was the strongest, indicating an important role of that enzyme in the adaptation to coal ash pollutants. The results of this study suggest that the modulation of the studied biochemical parameters in plants growing on coal ash is primarily species-dependent.

In the environment co-contamination of several toxicants commonly occurs. However, toxicological studies usually are focused on only one toxicant. The aim of this study was to investigate toxicity of silver nanoparticles (AgNP) and mycotoxin fumonisin B1 (FB1) and their possible interactions as well as to explore tentative mechanism of their toxic effect. Duckweed (Lemna minor L.) was treated with AgNP or FB1 (at concentrations 0.5 and 1.0 mg L−1) or with their combination at same concentrations for 3 days. Both AgNP and FB1, applied individually significantly affected levels of certain nutrients, reduced growth rate and the levels of photosynthetic pigments though AgNP at a much greater extent compared to FB1. Furthermore, AgNP induced ROS generation, lipid peroxidation and increase of antioxidative enzymes activities, while FB1 induced changes only in the activities of antioxidative enzymes. Those results implicate that phytotoxicity of both AgNP and FB1 can be associated with imbalance of mineral and cell redox status. However, toxic actions of AgNp singly applied were more pronounced. Combined treatment with AgNP and FB1 produced higher degree of changes in all parameters than corresponding concentrations of AgNP or FB1 alone implying their additive effects. Additionally, higher level of FB1 found in medium, and higher level of intracellular Ag following combined treatment indicates interaction of two toxicants at the transport level/uptake in the cell which resulted in higher accumulation of Ag in duckweed cells. The latter in turn exerted higher toxicity to duckweed compared to single treatment of AgNP.

To address and to compare the respective impact of gold and silver nanoparticles (Au and Ag NPs) in soil invertebrate, the earthworm Eisenia fetida was exposed to soil containing 2, 10, and 50 mg/kg of Au and Ag in both nanoparticulate and ionic forms for 10 days. Both metal NPs were 2–15 times less bioavailable than their ionic forms, and displayed similar transfer coefficients from soil to earthworm tissues. Both metal NPs triggered the onset of an oxidative stress as illustrated by increased glutathione S-transferase levels, decreased catalase levels, and increased malondialdehyde concentrations. Protein carbonylation distinguished the nanoparticular from the ionic forms as its increase was observed only after exposure to the highest concentration of both metal NPs. Au and Ag NPs triggered DNA modifications even at the lowest concentration, and both repressed the expression of genes involved in the general defense and stress response at high concentrations as did their ionic counterparts. Despite the fact that both metal NPs were less bioavailable than their ionic forms, at equivalent concentrations accumulated within earthworms tissues they exerted equal or higher toxic potential than their ionic counterparts. Capsule: At equivalent concentrations accumulated within earthworm tissues Au and Ag NPs exert equal or higher toxic potential than their ionic forms.

Interactions of two newly synthesized and six previously reported benzoxanthene lignans (BXLs), analogues of rare natural products, with DNA/RNA, G-quadruplex and HSA were evaluated by a set of spectrophotometric methods. Presence/absence of methoxy and hydroxy groups on the benzoxanthene core and minor modifications at C-1/C-2 side pendants – presence/absence of phenyl ring and presence/absence of methoxy and hydroxy groups on phenyl ring – influenced the fluorescence changes and the binding strength to double- stranded (ds-) and G-quadruplex structures. In general, compounds without phenyl ring showed stronger fluorescence changes upon binding than phenyl-substituted BXLs. On the other hand, BXLs with an unsubstituted phenyl ring showed the best stabilization effects of G-quadruplex. Circular dichroism spectroscopy results suggest mixed binding mode, groove binding and partial intercalation, to ds-DNA/RNA and end- stacking to top or bottom G-tetrads as the main binding modes of BXLs to those targets. All compounds exhibited micromolar binding affinities toward HSA and an increased protein thermal stability. Moderate to strong antiradical scavenging activity was observed for all BXLs with hydroxy groups at C-6, C-9 and C-10 positions of the benzoxanthene core, except for derivative bearing methoxy groups at these positions. BXLs with unsubstituted or low-substituted phenyl ring and one derivative without phenyl ring showed strong growth inhibition of Gram- positive Staphylococcus aureus. All compounds showed moderate to strong tumor cell growth- inhibitory activity and cytotoxicity.

This study focused on the short-term whole organism bioassays (WOBs) on fish (Danio rerio) and crustaceans (Gammarus fossarum and Daphnia magna) to assess the negative biological effects of water from the major European River Sava and the comparison of the obtained results with in vitro toxicity data (ToxCast database) and Risk Quotient (RQ) methodology. Pollution profiles of five sampling sites along the River Sava were assessed by simultaneous chemical analysis of 562 organic contaminants (OCs) of which 476 were detected. At each sampling site, pharmaceuticals/illicit drugs category was mostly represented by their cumulative concentration, followed by categories industrial chemicals, pesticides and hormones. An exposure-activity ratio (EAR) approach based on ToxCast data highlighted steroidal anti-inflammatory drugs, antibiotics, antiepileptics/neuroleptics, industrial chemicals and hormones as compounds with the highest biological potential. Summed EAR-based prediction of toxicity showed a good correlation with the estimated toxicity of assessed sampling sites using WOBs. WOBs did not exhibit increased mortality but caused various sub-lethal biological responses that were dependant relative to the sampling site pollution intensity as well as species sensitivity. Exposure of G. fossarum and D. magna to river water-induced lower feeding rates increased GST activity and TBARS levels. Zebrafish D. rerio embryo exhibited a significant decrease in heartbeat rate, failure in pigmentation formation, as well as inhibition of ABC transporters. Nuclear receptor activation was indicated as the biological target of greatest concern based on the EAR approach. A combined approach of short-term WOBs, with a special emphasis on sub-lethal endpoints, and chemical characterization of water samples compared against in vitro toxicity data from the ToxCast database and RQs can provide a comprehensive insight into the negative effect of pollutants on aquatic organisms.

The process of endosymbiosis is one of the promoters of speciation. The green hydra (Hydra viridissima Pallas, 1766) is a typical example of endosymbiosis. Its gastrodermal myoepithelial cells harbour the individuals of unicellular photoautotrophic algae. In this study we have developed healthy laboratory cultures of endosymbiotic green algae isolated from green hydra strains. The Hydra strains were collected from four different geographical localities, two of which were in Croatia (strains BV, T), one in Israel (strain M9) and one in Germany (strain HV). For the first time, endosymbiotic algae isolated from green hydra strains have been visualized and described by scanning and transmission electron microscopy. Isolated endosymbiotic algae were characterized by cytological morphometric parameters, enzyme activity and isoenzyme pattern analysis (catalase, peroxidase and esterase). In addition, cells of endosymbiotic algae were characterized by morphometric measurements of diameter, perimeter and area, and chloroplast area. Endosymbiotic algae HV (collected in Germany) and M9 (collected in Israel) were significantly different when compared with endosymbiotic algae T and BV collected in Croatia. The endosymbionts were more different with referent alga Parachlorella kessleri than with referent alga Chlorella vulgaris. The results obtained by isoenzyme pattern analysis also suggested that there was a difference between Croatian and European algal endosymbionts, i.e. the results indicate biological diversity among algal symbionts isolated from the green hydras from different geographical localities. Ultimately, five isoenzymes of catalase and five isoenzymes of peroxidase were resolved by PAGE electrophoresis. Catalase isoenzyme K1 appeared only in endosymbiotic alga M9 collected in Israel and catalase isoenzyme K3 only in endosymbiotic alga T collected in Croatia. Peroxidase isoenzyme P1 was observed only in endosymbiont HV while peroxidase isoenzyme P5 was observed in both endosymbionts HV and M9. Peroxidase isoenzymes P3 and P4 were visible in endosymbiotic algae BV and T collected in Croatia.

Airborne fly ash and related hazardous particles derived from coal combustion contaminate soil and groundwater, negatively affecting ecosystems. The aim of this study was chemical and toxicological evaluation of aqueous extracts of soil collected from the vicinity of a coal-fired Plomin power plant (PPP), using Lemna (Lemna minor L.) bioassay and additional biochemical indicators - photosynthetic pigments, lipid peroxidation, antioxidative enzymes and glutathione. Topsoil samples were collected from distances of 200, 300, 400 and 800 m from the PPP in accordance with the prevailing SW wind direction. Elevated levels of polycyclic aromatic hydrocarbons (up to 15,765 ng L−1) and potentially toxic trace elements were detected in the Plomin soil extracts (PEs) in comparison to control soil extract (CE). Trace elements accumulated in L. minor were mostly in accordance with their concentrations in PEs. The results demonstrate that PEs induced significant growth inhibition and other phytotoxic effects. Those effects can be related to damage caused by increased production of reactive oxygen species and impaired antioxidant levels. The connection among the phytotoxicity, a distribution of analyzed contaminants, and distances from the PPP is clearly established.

Introduction The phytochemical composition and biological activity of non-volatile components of Centaurea ragusina L. has not been studied previously. Objectives Our aim was to evaluate the phytochemical and bioactive potential (including interactions with polynucleotides) of C. ragusina L. depending on the origin of plant material (in vivo – leaves from natural habitats, ex vitro – leaves from plants acclimated from culture media, in vitro – leaves and calli from plants grown in culture media) and polarity of solvents used in extract preparation (80 and 96% ethanol and water combinations or single solvents). Methodology The polyphenol composition was determined by spectrophotometric and HPLC analysis. Biological activity of extracts was evaluated by following methods: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods for antioxidative activity, 2,3,5-triphenyl tetrazolium chloride (TTC) microdilution method for antibacterial activity, crystal-violet test for cytotoxic activity and thermal denaturation (TD) and circular dichroism (CD) for DNA/RNA interactions. Results Conditions for the most efficient polyphenol extraction were determined: the 80% ethanol/water solvent system was the most suitable for callus and leaf ex vitro samples and 80 or 96% ethanol for leaf in vivo samples. Significantly higher levels of chlorogenic acid and naringenin were detected in callus tissue than in vivo plant. Ethanolic extracts exhibited the significant antibacterial activity against Staphylococcus aureus ATCC 25923. DNA/RNA active compounds in plant extracts were detected by TD and CD methods. Conclusions Callus tissue and ex vitro leaves represent a valuable source of polyphenols as in vivo leaves. TD and CD can be applied for detection of DNA/RNA active compounds in extracts from natural resources. Copyright © 2017 John Wiley & Sons, Ltd.

The complexity of seasonally and spatially variable environments, coupled with complex biological interactions, makes it difficult to pinpoint biological responses to specific environmental stressors, including chemical pollution. To disentangle causative factors and reveal biomarker responses, we applied biomarker- based multivariate approaches to 15 native populations of Mediterranean mussel Mytilus galloprovincialis in spring and autumn. In addition, we used a subset of these populations in transplant experiments between clean and polluted environments in nature and in lab mesocosms. The extent of biomarker responses in native populations is affected by season, and significantly lower variability across seasons was observed among mussels from clean than from polluted sites. Results of paired block designed transplant experiment demonstrated both regional and pollution effect, with mussels uniformly exhibiting higher responses on more impacted sites in each of the Adriatic regions. Biomarker status of mussels varied among Adriatic regions in dependence on the set of environmental variables, and between clean and polluted sites in dependence on measured concentrations of metals in mussels' tissue. Results of the mesocosm experiment revealed distinctive biomarker responses of two populations of different origin when exposed to common conditions. Multivariate description of biomarker activity and application of specific experiments allowed us to link environmental condition, exposure to pollution and seasonality to mussels' biomarker responses.

To address and to compare the respective impact of gold and silver nanoparticles (Au and Ag NPs) in soil invertebrate, the earthworm

Allium-test je jednostavni biotest za određivanje ukupne toksičnosti i genotoksičnosti kojeg učenici uz pomoć nastavnika ili samostalno mogu provesti u školi te njime testirati učinak različitih otopina na modelni organizam Allium cepa L. (obični luk). Allium-test je dobar primjer projekta u nastavi jer se svojim opsegom može prilagoditi dobi učenika, a potiče njihovu kreativnost i samostalnost u provedbi i prezentiranju samog projekta. Sam projekt i rezultati istog potiču učenike na razvijanje svijesti o vodenom okolišu i učinku onečišćenja na svim njegovim razinama., Allium-test is a simple biotest for determining toxicity and genotoxicity which students can perform at school, with the help of teachers or independently, to test the effects of different solutions on the model organism Allium cepa L. (the common onion). The Allium test is a good example of project-based learning because it can be adapted to the age of the students, and it enforces their creativity and independence in the implementation and presentation of the project itself. The project itself and its results encourages students to develop awareness of the aquatic environment and the impact of pollution at all its levels.

Owing to its proven toxicity and mutagenicity, arsenic is regarded a principal pollutant in water used for drinking. The objective of this study was the toxicological and chemical evaluation of groundwater samples obtained from arsenic enriched drinking water wells before and after electrochemical and ozone-UV-H2O2- based advanced oxidation processes (EAOP). For this purpose, acute toxicity test with Daphnia magna and chronic toxicity test with Lemna minor L. were employed as well as in vitro bioassays using human peripheral blood lymphocytes (HPBLs). Several oxidative stress parameters were estimated in L.minor. Physicochemical analysis showed that EAOP treatment was highly efficient in arsenic but also in ammonia and organic compound removal from contaminated groundwater. Untreated groundwater caused only slight toxicity to HPBLs and D. magna in acute experiments. However, 7-day exposure of L. minor to raw groundwater elicited genotoxicity, a significant growth inhibition and oxidative stress injury. The observed genotoxicity and toxicity of raw groundwater samples was almost completely eliminated by EAOP treatment. Generally, the results obtained with L. minor were in agreement with those obtained in the chemical analysis suggesting the sensitivity of the model organism in monitoring of arsenic- contaminated groundwater. In parallel to chemical analysis, the implementation of chronic toxicity bioassays in a battery is recommended in the assessment of the toxic and genotoxic potential of such complex mixtures.

Drought is one of the major abiotic stresses affecting the productivity of Brassica crops. To understand the role of phytohormones in drought tolerance, we subjected Chinese cabbage (B. rapa ssp. pekinensis), white cabbage (B. oleracea var. capitata), and kale (B. oleracea var. acephala) to drought and examined the stress response on the physiological, biochemical and hormonal levels. The phytohormones abscisic acid (ABA), auxin indole-3-acetic acid (IAA), brassinosteroids (BRs), cytokinins (CKs), jasmonates (JAs), and salicylic acid (SA) were analyzed by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). Based on the physiological and biochemical markers the Chinese cabbage exhibited the lowest tolerance, followed by the white cabbage, while the kale appeared to be the most tolerant to drought. The drought tolerance of the kale correlated with increased levels of SA, ABA, IAA, CKs iP(R) and cZ(R), and typhasterol (TY), a precursor of active BRs. In contrast, the drought sensitivity of the Chinese cabbage correlated with a significant increase in ABA, JAs and the active BRs castasterol (CS) and brassinolide (BL). The moderately tolerant white cabbage, positioned between the kale and Chinese cabbage, showed more similarity in terms of the phytohormone patterns with the kale. We concluded that the drought tolerance in Brassicaceae is mostly determined by the increased endogenous levels of IAA, CKs, ABA and SA and the decreased levels of active BRs.

The endemic Croatian species Centaurea ragusina L., like other species from the genus Centaurea, has been traditionally used in Croatia as an antibacterial agent and for the treatment of gastrointestinal and urogenital disorders. In several chromatographic steps, three flavonoids and three sesquiterpene lactones (STLs) were isolated and identified from the most active fractions of the ethanol extract. Two STLs, one for which we created the trivial name ragusinin, and hemistepsin A are here reported for the first time as constituents of the genus Centaurea. All six compounds were screened for their effect on several tumor and one normal cell lines. Among them, ragusinin showed the best bioactivity and high specificity to affect tumor murine SCCVII, human HeLa and Caco-2 cell lines, but not the viability of normal V79 fibroblasts. Due to these characteristics the action of ragusinin was investigated in more detail. Since DNA is the primary target for many drugs with antibacterial and anticancer activity, we studied its interaction with ragusinin. Rather moderate binding affinity to DNA excluded it as the primary target of ragusinin. Due to the possibility of STL interaction with glutathione (GSH), the ubiquitous peptide that traps reactive compounds and other xenobiotics to prevent damage to vital proteins and nucleic acids, its role in deactivation of ragusinin was evaluated. Addition of the GSH precursor N- acetyl-cysteine potentiated the viability of HeLa cells, while the addition of GSH inhibitor L-buthionine sulfoximine decreased it. Moreover, pre-treatment of HeLa cells with the inhibitor of glutathione-S-transferase decreased their viability indicating the detoxifying role of GSH in ragusinin treated cells. Cell death, derived by an accumulation of cells in a G2 phase of the cell cylce, was shown to be independent of poly (ADP-ribose) polymerase and caspase-3 cleavage pointing toward an alternative cell death pathway.

Salinity is one of major abiotic stresses affecting Brassica crop production. Here we present investigations into the physiological, biochemical, and hormonal components of the short-term salinity stress response in Chinese cabbage seedlings, with particular emphasis on the biosynthesis and metabolism of auxin indole-3-acetic acid (IAA). Upon salinity treatments (50- 200 mM NaCl) IAA level was elevated in a dose dependent manner reaching 1.3-fold increase at the most severe salt treatment in comparison to the control. IAA precursor profiling suggested that salinity activated the indole-3-acetamide and indole-3-acetaldoxime biosynthetic pathways while suppressing the indole-3-pyruvic acid pathway. Levels of the IAA catabolites 2-oxoindole-3-acetic acid and indole-3-acetic acid-aspartate increased 1.7- and 2.0-fold, respectively, under the most severe treatment, in parallel with those of IAA. Conversely, levels of the ester conjugate indole-3-acetyl-1-O-s-d-glucose and its catabolite 2-oxoindole-3-acetyl-1-O-s-d-glucose decreased 2.5- and 7.0-fold, respectively. The concentrations of stress hormones including jasmonic acid and jasmonoyl-isoleucine (JA and JA-Ile), salicylic acid (SA) and abscisic acid (ABA) confirmed the stress induced by salt treatment: levels of JA and JA-Ile increased strongly under the mildest treatment, ABA only increased under the most severe treatment, and SA levels decreased dose-dependently. These hormonal changes were related to the observed changes in biochemical stress markers upon salt treatments: reductions in seedling fresh weight and root growth, decreased photosynthesis rate, increased levels of reactive oxygen species, and elevated proline content and the Na+/K+ ratio. Correlations among auxin profile and biochemical stress markers were discussed based on Pearson’s coefficients and principal component analysis (PCA).

Malondialdehyde (MDA) is stabile product of lipid peroxidation (LPO), and therefore MDA is frequently used as a biomarker of LPO. To determine MDA level in various biological samples (human plasma, fish liver tissue and cells in culture), we used an HPLC method with fluorescent detection based on 2-thiobarbituric acid (TBA) assay. The method was validated by the use of spiked pooled plasma samples. In tested concentration range (0.15–3.0 µmol/L) the method was linear (R2 = 0.9963), the between-day variability (coefficient of variations, CVs) was between 4.7 and 7.6%, the within-day variability CVs was between 2.6 and 6.4% and recovery was between 91.2 and 107.6%. The level of MDA in human plasma (healthy male, non-smokers, 46.3 ± 4.7 years; N = 38) was 2.2 ± 1.4 µmol/L; that in liver tissue of common carp (Cyprinus carpio; N = 12) was 0.02 ± 0.004 µmol/g tissue, and in cultured cells (human laryngeal carcinoma cells; N = 10) it was 0.18 ± 0.02 nmol/mg proteins. The HPLC-FL method is rapid, accurate and reliable to follow the extent of LPO in various biological samples, particularly in samples in which a low level of MDA is expected, such as cells in culture. Owing to the rapid analytical process and run time, it can be used for routine analysis of MDA in clinical laboratory. Copyright © 2014 John Wiley & Sons, Ltd.

Summary Leachates from active and closed municipal solid waste landfills can be a major source of contamination to groundwater and surface waters. In the present study the toxic and genotoxic potential of leachate from an old sanitary landfill prior to and following chemical and electrochemical treatments were assessed using Lemna, Allium, and comet tests. Photosynthetic pigments, malondialdehyde (indicator of lipid peroxidation) and antioxidant enzyme activities were evaluated as additional indicators of toxicity in duckweed. Following duckweed exposure to 25 % dilution of landfill leachate, growth rate and photosynthetic pigments content significantly decreased while lipid peroxidation increased despite stimulation of antioxidative defence mechanisms. Diluted leachate induced DNA strand breaks in duckweed cells as evidenced by the comet assay. Regarding the Allium test, untreated leachate caused inhibition of Allium cepa cell division and induction of mitotic and chromosomal aberrations. Although both water treatments completely reduced genotoxicity of leachate, the electrochemical method was found to be more efficient in removing toxic substances present in landfill leachate and thus more suitable for treating such leachates prior to their discharge into the environment. As landfill leachates pose a risk to human health and environment in general due to their (geno)toxicity, the present study demonstrates that the ecotoxicity/genotoxicity assays should be used in leachate risk assessment together with physicochemical analysis.

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.

Drought is one of the major abiotic stresses affecting the productivity of Brassica crops. To understand the role of phytohormones in drought tolerance, we subjected Chinese cabbage (B. rapa ssp. pekinensis), white cabbage (B. oleracea var. capitata), and kale (B. oleracea var. acephala) to drought and examined the stress response on the physiological, biochemical and hormonal levels. The phytohormones abscisic acid (ABA), auxin indole-3-acetic acid (IAA), brassinosteroids (BRs), cytokinins (CKs), jasmonates (JAs), and salicylic acid (SA) were analyzed by ultra-high-performance liquid chromatography&ndash, tandem mass spectrometry (UHPLC-MS/MS). Based on the physiological and biochemical markers the Chinese cabbage exhibited the lowest tolerance, followed by the white cabbage, while the kale appeared to be the most tolerant to drought. The drought tolerance of the kale correlated with increased levels of SA, ABA, IAA, CKs iP(R) and cZ(R), and typhasterol (TY), a precursor of active BRs. In contrast, the drought sensitivity of the Chinese cabbage correlated with a significant increase in ABA, JAs and the active BRs castasterol (CS) and brassinolide (BL). The moderately tolerant white cabbage, positioned between the kale and Chinese cabbage, showed more similarity in terms of the phytohormone patterns with the kale. We concluded that the drought tolerance in Brassicaceae is mostly determined by the increased endogenous levels of IAA, CKs, ABA and SA and the decreased levels of active BRs.

Screening for mutagens in environmental complex mixtures, such as surface water or industrial wastewater, is gradually being accepted as a routine methodology in monitoring program. In the present work, the simplified A. cepa root assay was employed in order to evaluate the possible cyto- and genotoxic effects of surface and wastewaters collected along or near the Sava River (Croatia) over a 3-month monitoring period. Physicochemical characterization of the water pollution was determined by measuring 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 polluted water samples (industrial effluents) caused an inhibition of root growth over 50%, a decrease of mitotic index over 40% and considerable increase of chromosomal aberrations in comparison to control. The measured biological effects of some water samples appeared related to the physicochemical characteristics. Therefore, together with the conventional chemical analysis, mutagenicity/genotoxicity assays should be included as complementary parameters in water quality monitoring programs. Their use could permit the quantification of mutagenic hazards in surface and wastewaters.

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 the individuals of unicellular green algae. Morphological characteristics of isolated algae determined by light and electron microscopy were presented. Cytological morphometric parameters (cell area, cell radius, chloroplast area) of isolated alga from green hydra (Cx), as well as referent species Chlorella kessleri (Ck) and Chlorella vulgaris (Cv), revealed the similarity between the isolated endosymbionts 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 specifically expressed in 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.

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 three 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.

In the present study toxicity of Frangula alnus Mill. bark, widely used as laxative, was investigated. Human peripheral blood lymphocytes (HPBLs) were treated with F. alnus bark extract or emodin (emodin is bark component with laxative property), and cytotoxicity, genotoxicity and parameters of oxidative stress were assessed. Also, polyphenol content of bark extract and antioxidant activity of the extract and emodin measured by DPPH, ABTS and FRAP methods were examined. The bark extract (500 μg/ml) produced cell death and DNA damage, while level of ROS changed at 250 μg/ml. Emodin induced cell death and DNA damage at 150 μg/ml and 200 μg/ml, respectively, and the increase of ROS was observed at 25 μg/ml. These results suggest that both, bark extract and emodin, are cyto/genotoxic to HPBLs and that oxidative stress is involved in the mechanism of their toxicity. The results on antioxidant activity showed that, unlike emodin, bark extract possess moderate antioxidant capacity (44.6%, 46.8% and 2.25 mmol Fe(2+)/g measured by DPPH, ABTS and FRAP assay, respectively) that can be related to relatively high phenolic content (116.07 mg/g). However, due to toxicological properties use of F. alnus bark as well as emodin-containing preparations should be taken with caution.

Malondialdehyde (MDA) is stabile product of lipid peroxidation (LPO), and therefore MDA is frequently used as a biomarker of LPO. To determine MDA level in various biological samples (human plasma, fish liver tissue and cells in culture), we used an HPLC method with fluorescent detection based on 2-thiobarbituric acid (TBA) assay. The method was validated by the use of spiked pooled plasma samples. In tested concentration range (0.15-3.0 µmol/L) the method was linear (R(2) = 0.9963), the between-day variability (coefficient of variations, CVs) was between 4.7 and 7.6%, the within-day variability CVs was between 2.6 and 6.4% and recovery was between 91.2 and 107.6%. The level of MDA in human plasma (healthy male, non-smokers, 46.3 ± 4.7 years; N = 38) was 2.2 ± 1.4 µmol/L; that in liver tissue of common carp (Cyprinus carpio; N = 12) was 0.02 ± 0.004 µmol/g tissue, and in cultured cells (human laryngeal carcinoma cells; N = 10) it was 0.18 ± 0.02 nmol/mg proteins. The HPLC-FL method is rapid, accurate and reliable to follow the extent of LPO in various biological samples, particularly in samples in which a low level of MDA is expected, such as cells in culture. Owing to the rapid analytical process and run time, it can be used for routine analysis of MDA in clinical laboratory.

Leachates from active and closed municipal solid waste landfills can be a major source of contamination to groundwater and surface waters. In the present study the toxic and genotoxic potential of leachate from an old sanitary landfill prior to and following chemical and electrochemical treatments were assessed using Lemna, Allium, and comet tests. Photosynthetic pigments, malondialdehyde (indicator of lipid peroxidation) and antioxidant enzyme activities were evaluated as additional indicators of toxicity in duckweed. Following duckweed exposure to 25 % dilution of landfill leachate, growth rate and photosynthetic pigments content significantly decreased while lipid peroxidation increased despite stimulation of antioxidative defence mechanisms. Diluted leachate induced DNA strand breaks in duckweed cells as evidenced by the comet assay. Regarding the Allium test, untreated leachate caused inhibition of Allium cepa cell division and induction of mitotic and chromosomal aberrations. Although both water treatments completely reduced genotoxicity of leachate, the electrochemical method was found to be more efficient in removing toxic substances present in landfill leachate and thus more suitable for treating such leachates prior to their discharge into the environment. As landfill leachates pose a risk to human health and environment in general due to their (geno)toxicity, the present study demonstrates that the ecotoxicity/genotoxicity assays should be used in leachate risk assessment together with physicochemical analysis., Procjedne vode s aktivnih i zatvorenih odlagališta krupnog otpada mogu biti glavni izvor onečišćenja podzemnih i površinskih voda. U ovom su radu putem Lemna-, Allium- i komet-testa procijenjeni toksični i genotoksični učinci procjedne vode prikupljene sa starog odlagališta otpada Piškornica (Koprivnica) prije i nakon kemijskog ili elektrokemijskog pročišćavanja. Dodatni pokazatelji toksičnosti praćeni u vodenoj leći bili su sadržaj fotosintetskih pigmenata i malondialdehida (indikator lipidne peroksidacije) te aktivnosti antioksidacijskih enzima. U toj je biljci 25 %-tno razrjeđenje procjedne vode uzrokovalo smanjenje rasta i sadržaja pigmenata dok je lipidna peroksidacija porasla unatoč stimulaciji antioksidacijskih obrambenih mehanizama. Komet testom je utvrđeno oštećenje molekule DNA u stanicama vodene leće. Glede Alium testa, nepročišćena procjedna voda uzrokovala je inhibiciju diobe u luku (Allium cepa) te pojavu mitotskih i kromosomskih aberacija. Iako su obje metode pročišćavanja bile vrlo učinkovite u uklanjanju genotoksičnih tvari prisutnih u odlagališnom eluatu, elektrokemijska metoda pokazala se učinkovitija u uklanjanju toksičnih tvari iz eluata, te time i pogodnija za obradu takvih vrsta eluata prije njihova ispuštanja u okoliš. Kako su odlagališni eluati zbog svoje genotoksičnosti opasni za ljudsko zdravlje i okoliš općenito, rezultati ovog istraživanja pokazuju da bi se testovi toksičnosti i genotoksičnosti trebali upotrebljavati u procjeni rizika odlagališnih eluata, zajedno s fizikalno-kemijskom analizom.

Water deficit in the soil leads to osmotic stress in plants. The type of stress affects plant water relations, osmolyte accumulation and oxidative stress balance. The present study aimed to investigate the effects of osmotic stress on the Croatian perennial species Fibigia triquetra (DC.) Boiss, adapted to a hot and dry habitat. Plants grown in culture conditions were subjected to isoosmotic concentrations of mannitol and polyethylene glycol (PEG) and certain physiological and oxidative stress parameters were analyzed during a period of 14 days. Dry weight and proline content in Fibigia triquetra shoots increased in response to osmotic stress while the relative water content decreased. After an initial rise, chlorophyll and carotenoid levels in treated plants dropped to untreated plant levels. Oxidative damage to proteins and especially to lipids was evident upon PEG-induced osmotic stress. Superoxide dismutase and ascorbate peroxidase appear to play an essential protective role in stressed plants. Regardless of the osmotic agent, accumulation of heatshock proteins of 70 kDa was noticed under osmotic stress. The tolerance of the plant species to osmotic stress seems to be associated with increased capacity of the antioxidative system and efficient photoprotective system. Normal 0 21 false false false HR X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin;}

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.

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.

Summary The present study investigated the effects of salinity on the perennial species Centaurea ragusina L. interesting as a potential cash crop plant. Plants grown in culture conditions were subjected to increasing salt (0–600 mM NaCl) or mannitol (300 mM) treatments for two weeks. Effects of isoosmotic concentrations of NaCl (150 mM) and mannitol were compared and discussed in order to discriminate possible differences in C. ragusina response to ionic (NaCl) and osmotic (mannitol) components of salinity. C. ragusina plants used Na and to a lesser extent Cl ions as a primary osmotica though with higher salinity, proline accumulation increased as well. Concurrently, with increasing salinity significant reductions in plant K, Mg and Ca concentrations occurred. In addition, lower salt concentrations induced leaf succulence and increased leaf relative water content (RWC). A threshold salinity above which C.ragusina L. showed signs of damage and growth inhibition was reached at 300 mM. Activities of superoxide dismutase, catalase and ascorbate peroxidase in salinized plants seem to play an essential protective role in the scavenging processes. Regardless of the high induction of antioxidative system and massive proline accumulation, mannitol caused decrease of RWC and oxidative damage to proteins and lipids. Considering the abundance of some photosynthetic proteins (Rubisco, D1, LHCI, LHII and FNR) and PSII efficiency, it can be concluded that both salt and mannitol impaired photosynthesis in C. ragusina though salt to a much lesser extent. The results suggest that the major reason for the particular threshold of salinity tolerance in C. ragusina can be attributed to limited dilution capacity of succulent tissue. The tolerance strategies of C. ragusina to moderate salinity seem to include osmotic adjustment achieved through salt ions uptake as a dominant strategy but also highly inducible antioxidative defense.

In this research, toxicological safety of two newly developed methods for the treatment of landfill leachate from the Piskornica (Croatia) sanitary landfill was investigated. Chemical treatment procedure combined chemical precipitation with CaO followed by coagulation with ferric chloride and final adsorption by clinoptilolite. Electrochemical treatment approach included pretreatment with ozone followed by electrooxidation/electrocoagulation and final polishing by microwave irradiation. Cell viability of untreated/treated landfill leachate was examined using fluorescence microscopy. Cytotoxic effect of the original leachate was obtained for both exposure periods (4 and 24 h) while treated samples showed no cytotoxic effect even after prolonged exposure time. The potential DNA damage of the untreated/treated landfill leachate was evaluated by the comet assay and cytokinesis-block micronucleus (CBMN) assay using either human or plant cells. The original leachate exhibited significantly higher comet assay parameters compared to negative control after 24 h exposure. On the contrary, there was no significant difference between negative control and chemically/electrochemically treated leachate for any of the parameters tested. There was also no significant increase in either CBMN assay parameter compared to the negative control following the exposure of the lymphocytes to the chemically or electrochemically treated landfill leachate for both exposure periods while the original sample showed significantly higher number of micronuclei, nucleoplasmic bridges and nuclear buds for both exposure times. Results suggest that both methods are suitable for the treatment of such complex waste effluent due to high removal efficiency of all measured parameters and toxicological safety of the treated effluent.

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.