Does exposure to weathered coal ash with an enhanced content of uranium-series radionuclides affect flora? Changes in the physiological indicators of five referent plant species.

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.

Assessment of Metal Intake by Selected Food Supplements Based on Beehive Products.

The aim of this study was to determine the quantity of particular toxic metals (Pb, Cd, As, Hg) and micronutrients (Cr, Fe, Co, Ni, Cu, Zn, Se) in the recommended daily dose of 51 food supplements based on beehive products. Samples taken from the Croatian market were submitted for the identification/quantification of studied metals and micronutrients. It was carried out by means of inductively coupled plasma mass spectrometry (ICP-MS). Eleven samples (21.57%) showed an increased concentration of total arsenic, three samples (5.88%) contained an increased concentration of total iron, and eight samples (15.68%) had an increased concentration of total nickel. Three samples (5.88%) contained an increased concentration of zinc, while one sample (1.96%) contained an increased concentration of selenium. Increased levels of certain toxic metals and micronutrients do not pose a danger to human health because the amount identified was less than what can cause toxic effects in humans. All other analysed metals and micronutrients fell within the defined literature values. Despite certain increases in particular parameters, all samples met the established toxicity criteria. This study evidenced their safety if consumed in the recommended daily dose.

Assessment of intracellular accumulation of cadmium and thallium.

The aim of the study was to develop fast and accurate method for assessment of intracellular level of cadmium (Cd) and thallium (Tl), and to establish accumulation of the metals in the cells. HepG2 cells were treated with Cd or Tl (1.0 or 10.0 mg/L; 24 h) and level of Cd or Tl was assessed. ICP-MS was applied and the method was optimized and validated. Correlation coefficient (R2) for Cd was 0.9999 with intercept 0.0732 while for Tl was 1.00009 with intercept -0.1497, and limit of detection (LOD) for Cd was 0.020 µg/L and for Tl 0.097 µg/L. Both metals, Cd and Tl, accumulate in the cells in concentration-dependent manner. However, higher uptake of Cd in comparison to Tl was observed. Cells treated with the same concentration of the metal (1.0 mg/L) accumulated 10.0% of Cd and 1.0% of Tl. Higher uptake of Cd than Tl can explain higher toxicity of Cd toward HepG2 cells. Obtained results imply to the importance of monitoring the level of metals in the cells in order to connect changes at the molecular level with exposure to specific metal.

VERIFICATION OF AUTHENTICITY OF GINKGO BILOBA L. LEAF EXTRACT AND ITS PRODUCTS PRESENT ON THE CROATIAN MARKET BY ANALYSIS OF QUANTITY AND RATIO OF GINKGO FLAVONE GLYCOSIDES (QUERCETIN, KAEMPFEROL AND ISORHAMNETIN) TO TERPENE TRILACTONES TO THE EFFECT OF UNMASKING COUNTERFEIT DRUGS ENDANGERING PATIENT HEALTH.

Ginkgo biloba L. is the eldest plant growing on the Earth; preparations made of its leaves and seeds represent an integral part of the Chinese medicine for over a millennium. The plant species was first discovered by Linnaeus in 1771, its name thereby originating from the Latin words bis (two) and lobus (lobe), which duly illustrate the specific shape of its leaf. Contemporary Ginkgo biloba L. plant based pharmaceuticals mostly comprise extracts recovered from leaves harvested during fall, when the concentration of active components reaches its peak. Recent investigations have managed to establish the chemical composition of the plant leaf, together with the mechanisms underlying its beneficial effects on rheological profile of the blood and acceleration of its flow. High price of these preparations and their vast popularity have soon become an incentive for counterfeiting Ginkgo biloba L. extracts and the release of bogus drugs comprising cheaper extracts coming from other plants. Namely, modern Ginkgo biloba L.-based medicinal products and food supplements comprise extracts recovered from the plant leaf that get to be standardized according to its key pharmacological active components, most often flavone glycosides (represented in the share of 22%-27%) and terpene trilactones (represented in the share of 6%-7%). The flavonoids that predominate such preparations and are most relevant from the pharmacological standpoint are quercetin, kaempferol and isorhamnetin, their total amount and mutual ratios, thereby being an unquestionable indicator of the extract authenticity. Therefore, most of the analyses aiming at verifying the authenticity of a given Ginkgo biloba L.-based product boil down to the analysis of these parameters. Counterfeiting involves partial or full replacement of the Ginkgo biloba L. extract (GBE) with a cheaper plant extract of a similar composition, the latter occasionally being enriched with an additional amount of flavonoids, most often quercetin, not originating from the Ginkgo biloba L. plant. The aim of this study was to verify the authenticity and quality of Ginkgo biloba L.-based products circulating on the Croatian market. To that effect, 10 samples of products produced by various manufacturers were analyzed in a certified laboratory. The parameters based on which the authenticity of the preparations was assessed were the shares of aglycones of typical ginkgo flavone glycosides, that is to say, quercetin, kaempferol and isorhamnetin, and mutual ratios of the established quantities of quercetin to kaempferol as the key clues to unmasking Ginkgo extracts counterfeiting. The amount of ginkgo flavone glycosides was established using high performance liquid chromatography. The analysis proved 80% of the samples analyzed to be conformant to the label statements as regards the total amount of flavone glycosides and their mutual ratios. In 20% of the samples, the ratio of quercetin to kaempferol deviated from normal values; on top of that, the presence of the phytoestrogen genistein, one of the components typically comprised by the Sophora japonica L. plant, was also proven, documenting counterfeiting of the GBE and its replacement by the Sophora japonica L. extracts in the samples under consideration. Due to the untrue label statements descriptive of these products, the information on the presence of pharmacologically active genistein was neglected to be mentioned despite its unfavorable health impact that can be expected in some consumer groups. The results of this study indicated the frequency of counterfeiting the Ginkgo biloba L.-based products found on the Croatian market to be deemed substantial. Therefore, a more rigorous and more thorough control of these products and sanctioning of irresponsible manufacturers and distributers is proposed, so as to contribute to a higher market representation of high-quality products, as well as to avoid health risks and downsize the rate of their counterfeiting.

The effect of hazardous pollutants from coal combustion activity: Phytotoxicity assessment of aqueous soil extracts.

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.

Removing aflatoxin M1 from milk with native lactic acid bacteria, centrifugation, and filtration.

In order to minimise human exposure to aflatoxin M1 (AFM1) the levels of this highly carcinogenic mycotoxin in milk, heat-treated milk, and other dairy products have been limited to <0.05 µg kg-1. However, its removal from dairy products presents a challenge for dairy producers, as commercial additives change organoleptic properties, and filtration alone yields poor results. The aim of this study was to find a strain of lactic acid bacteria (LAB) from milk or dairy products that most effectively binds AFM1 and to see whether heat treatment of the selected LAB affects the binding efficiency. We also wanted to investigate whether centrifugation can improve filtering of the obtained AFM1-LAB complexes from milk. To do that, we isolated and identified 10 native LAB species/strains, incubated their viable or heat-treated cells (108 CFU mL-1) in milk spiked with 0.5 µg L-1of AFM1 at 4 °C for 0, 2, 4, and 24 h, and quantified the amount of unbound AFM1 with HPLC. AFM1 binding efficiency ranged from 21 to 92 % for viable cells and from 26 to 94 % for the heattreated ones. Since both viable and heat-treated Lactobacillus plantarum KM showed the best results, we used it for the next step in AFM1 removal from milk. Heat treatment in combination with filtration and centrifugation yielded removal as high as 96 %.

PRESENCE OF CITRININ IN GRAINS AND ITS POSSIBLE HEALTH EFFECTS.

BACKGROUND: Citrinin is a mycotoxin produced by several species of the genera Aspergillus, Penicillium and Monascus and it occurs mainly in stored grain. Citrinin is generally formed after harvest and occurs mainly in stored grains, it also occurs in other plant products. Often, the co-occurrence with other mycotoxins is observed, especially ochratoxin A, which is usually associated with endemic nephropathy. At the European Union level, systematic monitoring of Citrinin in grains began with the aim of determining its highest permissible amount in food. Thus, far the systematic monitoring of the above mentioned mycotoxin in Croatia is yet to begin. MATERIALS AND METHODS: The main goal of this study was to determine the presence of Citrinin in grains sampled in the area of Medimurje, Osijek-Baranja, Vukovar-Srijem and Brod-Posavina County. For the purpose of identification and quantification of citrinin, high performance liquid chromatograph (HPLC) with fluorescence was used (Calibration curve k > 0.999; Intra assay CV = 2.1%; Inter assay CV = 4.3%; LOQ < 1 µg/kg). RESULTS: From the area of Medimurje County, 10 samples of corn and 10 samples of wheat were analyzed. None of the samples contained Citrinin (<1 µg/kg). From the area of Osijek-Baranja and Vukovar-Srijem County, 15 samples from each County were analyzed. The mean value for the samples of Osijek-Baranja County was 19.63 µg/kg (median=15.8 µg/kg), while for Vukovar-Srijem County the mean value of citrinin was 14,6 µg/kg (median=1.23 µg/kg). From 5 analyzed samples from Brod-Posavina County, one of the samples contained citrinin in the amount of 23.8 µg/kg, while the registered amounts in the other samples were <1 µg/kg. CONCLUSION: The results show that grains from several Counties contain certain amounts of Citrinin possibly indicating a significant intake of citrinin in humans. It must be stated that grains and grain-based products are the basis of everyday diet of all age groups, especially small children, where higher intake of citrinin can occur. Consequently, we emphasize the need for systematic analysis of larger amount of samples, from both large grains and small grains, especially in the area of Brod-Posavina County, in order to obtain more realistic notion of citrinin contamination of grains and to asses the health risk in humans.