Željka Vidaković-Cifrek

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 effect of oil industry high density brines on duckweed Lemna minor L.

Duckweed Lemna minor L. is a suitable plant model for toxicity evaluation of many substances due to its small size, rapid growth and ease of culture. Saturated water solutions of calcium chloride and calcium bromide and their 1:1 mixture are commonly used as “high density brines” for pressure control in oil wells. These solutions were added in Hoaglands nutrient medium in amounts appropriate to achieve 0.5%, 1.0%, 1.5% and 2.0% (v/v) dilutions and after two weeks of exposure the effect of tested chemicals on growth was estimated by counting fronds, measuring fresh and dry weights and determining total surface area of plants. Chlorophyll and carotenoid content in Lemna minor was also measured. Additionally, anthocyanin content in Spirodela polyrrhiza (L.) Schleiden was determined. During 14 days of exposure tested chemicals in lower concentrations (0.5%, 1.0% and 1.5% v/v) promoted the growth of Lemna minor, but they inhibited it in the highest (2.0% v/v). With increased concentration of tested solutions the concentrations of chlorophyll a and chlorophyll b were correspondingly higher in comparison with the control. Total carotenoid content and chl a/chl b ratio were also increased. The highest anthocyanin content in lower epidermis of Spirodela polyirhiza was noticed after the treatment with media containing 2.0% (v/v) CaCl2 and 1:1 mixture of CaCl2 and CaBr2, but lower concentrations of all three tested solutions also resulted in anthocyanin content increase.

Effect of Copper on the Toxicity and Genotoxicity of Cadmium in Duckweed (Lemna minor L.)

Effect of Copper on the Toxicity and Genotoxicity of Cadmium in Duckweed (Lemna Minor L.) We investigated interactions between copper (in the concentrations of 2.5 μmol L-1 and 5 μmol L-1) and cadmium (5 μmol L-1) in common duckweed (Lemna minor L.) by exposing it to either metal or to their combinations for four or seven days. Their uptake increased with time, but it was lower in plants treated with combinations of metals than in plants treated with either metal given alone. In separate treatments, either metal increased malondialdehyde (MDA) level and catalase and peroxidase activity. Both induced DNA damage, but copper did it only after 7 days of treatment. On day 4, the combination of cadmium and 5 μmol L-1 copper additionally increased MDA as well as catalase and peroxidase activity. In contrast, on day 7, MDA dropped in plants treated with combinations of metals, and especially with 2.5 μmol L-1 copper plus cadmium. In these plants, catalase activity was higher than in copper treated plants. Peroxidase activity increased after treatment with cadmium and 2.5 μmol L-1 copper but decreased in plants treated with cadmium and 5 μmol L-1 copper. Compared to copper alone, combinations of metals enhanced DNA damage after 4 days of treatment but it dropped on day 7. In conclusion, either metal given alone was toxic/genotoxic and caused oxidative stress. On day 4 of combined treatment, the higher copper concentration was more toxic than either metal alone. In contrast, on day 7 of combined treatment, the lower copper concentration showed lower oxidative and DNA damage. These complex interactions can not be explained by simple antagonism and/or synergism. Further studies should go in that direction. Učinak bakra na toksičnost i genotoksičnost kadmija u vodenoj leći (Lemna minor L.) U svrhu istraživanja interakcija između bakra kao esencijalnog elementa te kadmija kao neesencijalnog i toksičnog metala, vodenu leću Lemna minor L. uzgajali smo na podlogama s kadmijem (5 μmol L-1) odnosno s bakrom (2,5 μmol L-1 i 5 μmol L-1) te s njihovim kombinacijama. Unos metala u biljke povećavao se s trajanjem pokusa, a kod kombinacije metala u biljkama je izmjerena niža količina kadmija nego u onima uzgajanima samo na kadmiju. U biljkama tretiranim pojedinačnim metalom došlo je do povećanja sadržaja malondialdehida (MDA) te aktivnosti katalaze i peroksidaze u odnosu na kontrolne biljke. Također, primijećeno je oštećenje DNA iako kod bakra tek sedmog dana tretmana. Količina MDA i aktivnost obaju enzima dodatno se povećala na tretmanu kombinacijom kadmija i bakra (5 μmol L-1) nakon četvrtog dana pokusa, dok se količina MDA smanjila nakon sedmog dana kod kombinacije kadmija i 2,5 μmol L-1 bakra. U tim biljkama primijećena je i veća aktivnost katalaze, dok je aktivnost peroksidaze porasla na tretmanu kadmijem i 2,5 μmol L-1 bakrom, ali se smanjila na tretmanu kadmijem i 5 μmol L-1 bakrom. Oštećenje DNA koje je bilo veće kod kombinacije metala nakon četvrtog dana, osobito u usporedbi sa samim bakrom, smanjilo se nakon sedmog dana pokusa. Iz ovih rezultata može se zaključiti da su oba metala u istraživanim koncentracijama toksična i genotoksična za vodenu leću i da uzrokuju oksidacijski stres. Kadmij u kombinaciji s bakrom više koncentracije bio je toksičniji od pojedinačnih metala nakon četvrtog dana pokusa, dok su u biljaka tretiranih kombinacijom kadmija i bakra niže koncentracije toksični učinci bili manji. Budući da su primijećene interakcije vrlo kompleksne i ne uključuju samo antagonizam odnosno sinergizam potrebna su daljnja istraživanja.

In vitro conditions affect photosynthetic performance and crassulacean acid metabolism in Mammillaria gracilis Pfeiff. tissues

Mammillaria gracilis Pfeiff. plants cultivated in the pot (pot plants, PP), as well as in vitro-grown normal shoots (NS), habituated callus (HC), hyperhydric shoots (HS), and tumour tissue (TT) were investigated in order to reveal the influence of in vitro culture on functionality of the photosynthetic apparatus and CAM photosynthesis in cactus M. gracilis Pfeiff. Photosynthetic pigments content as well as maximum (Fv/Fm) and effective (ΦPSII) quantum yield of photosystem II (PSII) decreased in all in vitro-grown tissues in comparison to PP. The decrease observed in hyperhydric HC, HS and TT correlated with a low expression of Rubisco large subunit (RbcL) and β subunit of ATP synthase (β ATP synt) and almost undetectable levels of protein D1, light-harvesting chlorophyll a/b-binding protein (LHCII) and cytochrome f protein of thylakoid Cyt b6/f-complex (Cyt f) found in these tissues. As for crassulacean acid metabolism (CAM) pattern, PP and NS expressed diurnal acid fluctuation, while HC, HS and TT failed to show it. Nevertheless, all M. gracilis tissues exhibited diurnal changes of phosphoenolpyruvate carboxylase (PEPC) activity indicating the typical CAM physiology. In conclusion, the photosynthesis was down-regulated in all in vitro-grown tissues. NS maintained typical CAM photosynthesis, while HC, HS and TT withheld PEPC activity, but not acid accumulation specific for CAM. Minor changes observed in NS in comparison to PP could be attributed to the sugar supplementation while the more prominent deviations found in HC, HS and TT could be correlated with hyperhydricity and the loss of characteristic tissue organisation pattern.

Influence of digested wastewater sludge on early growth of the perennial ryegrass (Lolium perenne L.)

Effects of residual wastewater sludge, amended with zeolites, on the early growth of perennial ryegrass (Lolium perenne L.) were investigated. For that purpose growth parameters, content of photosynthetic pigments, substrate characteristics, levels of macro- and micronutrients, as well as toxic elements in plant tissue, plant inorganic matter and substrates, sewage sludge and soil, were determined. Among the growth indicators, only protein content and inorganic matter in plant tissue were found to be influenced by the type and composition of the substrate. Sewage sludge was found to contain elevated levels of Mo, Ni, Pb, Sb, Se, Sr and Zn, while Cu and Sn were found in an order of magnitude higher levels compared to the control sample. Application of sewage sludge, however, modified only the content of Cu and Zn in the plant tissue, inducing accumulation of these elements by a factor of 2. Despite the fact that all other measured elements (Al, As, Ba, Ca, Cd, Co, Cr, Cs, Fe, K, Li, Mg, Mn, Na, Ti, U and V) were found in plant tissue at similar level regardless of the substrate metal loading, accumulation indices point to overall lower bioavailability of elements from digested sludge in comparison with control substrate. Moreover, metal concentrations in all analyzed plant samples were below legally established limits.

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.

OIL AND GAS INDUSTRIAL CHEMICALS' CYTOTOXICITY STUDIED BY ALLIUM TEST

Allium test has already been used to determine cytotoxicity of waste drilling fluids. In the present work the cytotoxicity of four pure chemicals (Defoamex, Idthin 400, Magco Thin and Slick Pipe) was investigated. Those chemicals are components of drilling fluids, therefore, they are usually constituents of oil and gas industry waste waters. The tested chemicals were prepared in 1:9 dilution and cytotoxic effects on root-tip meristem ofAllium ascalonicum were analysed after 24-, 48- and 72-hour-treatments. All samples showed cytotoxicity which was proved by cytogenetic parameters such as inhibition of mitotic activity and increase of mitotic abnormalities and chromosomal aberrations in comparison with the control. Almost all mitotic abnormalities induced by four chemicals tested were the result of disturbed spindle mechanisms accompanied with stickiness. Slick Pipe revealed the most prominent mitodepressive effect and induced a high number of abnormalities. It caused a significant decrease of mitotic activity and increase of mitotic abnormalities after all three treatment durations, while chemicals Defoamex and Magco Thin showed a significant decrease of mitotic activity only after 72-hour-treatment. Chemicals Defoamex, Idthin 400 and Magco Thin revealed a significant increase of mitotic abnormalities after 48- and 72-hour-treatments.

Assessment of acrylamide toxicity using a battery of standardised bioassays

Acrylamide is a monomer widely used as an intermediate in the production of organic chemicals, e.g. polyacrylamides (PAMs). Since PAMs are low cost chemicals with applications in various industries and waste- and drinking water treatment, a certain amount of non-polymerised acrylamide is expected to end up in waterways. PAMs are non-toxic but acrylamide induces neurotoxic effects in humans and genotoxic, reproductive, and carcinogenic effects in laboratory animals. In order to evaluate the effect of acrylamide on freshwater organisms, bioassays were conducted on four species: algae Desmodesmus subspicatus and Pseudokirchneriella subcapitata, duckweed Lemna minor and water flea Daphnia magna according to ISO (International Organization for Standardisation) standardised methods. This approach ensures the evaluation of acrylamide toxicity on organisms with different levels of organisation and the comparability of results, and it examines the value of using a battery of low-cost standardised bioassays in the monitoring of pollution and contamination of aquatic ecosystems. These results showed that EC50 values were lower for Desmodesmus subspicatus and Pseudokirchneriella subcapitata than for Daphnia magna and Lemna minor, which suggests an increased sensitivity of algae to acrylamide. According to the toxic unit approach, the values estimated by the Lemna minor and Daphnia magna bioassays, classify acrylamide as slightly toxic (TU=0-1; Class 1). The results obtained from algal bioassays (Desmodesmus subspicatus and Pseudokirchneriella subcapitata) revealed the toxic effect of acrylamide (TU=1-10; Class 2) on these organisms. Akrilamid je monomer koji se koristi kao intermedijer u proizvodnji organskih kemikalija, npr. poliakrilamida (PAM-a). PAM se primjenjuje u različitim industrijama te u obradi otpadnih voda i tretmanu vode za piće, a prihvatljiv je i zbog niske cijene. Široka upotreba PAM-a u spomenute svrhe predstavlja rizik od toga da određena količina nepolimeriziranog akrilamida dospije u vodene ekosustave. PAM je netoksičan, ali akrilamid ima neurotoksične učinke u ljudi te je također dokazana njegova genotoksičnost, kancerogenost i štetan utjecaj na reprodukcijski sustav u laboratorijskih životinja. U cilju procjene toksičnosti akrilamida provedeni su biotestovi na četirima vrstama slatkovodnih organizama: na zelenim algama Desmodesmus subspicatus i Pseudokirchneriella subcapitata, vodenoj leći Lemna minor i vodenbuhi Daphnia magna. Biotestovi su provedeni prema standardiziranim ISO metodama, što osigurava procjenu toksičnosti akrilamida na organizme različitog stupnja organizacije, usporedivost rezultata i procjenu pogodnosti korištenja niza standardiziranih biotestova prihvatljive cijene za praćenje zagađenja i onečišćenja vodnih ekosustava. Rezultati su pokazali da su EC50 vrijednosti niže nakon izlaganja zelenih algi Desmodesmus subspicatus i Pseudokirchneriella subcapitata akrilamidu u odnosu na vrijednosti dobivene za vodenu leću Lemna minor i vodenbuhu Daphnia magna, što pokazuje veću osjetljivost algi. S obzirom na klasifikacijski sustav za izražavanje stupnja toksičnosti, a na temelju vrijednosti utvrđenih za vodenu leću Lemna minor i vodenbuhu Daphnia magna, akrilamid se može klasificirati kao slabo toksičan (TU=0-1; razred 1). Rezultati dobiveni testovima na algama (Desmodesmus subspicatus i Pseudokirchneriella subspicata) dokazuju toksičan učinak akrilamida na tim organizmima (TU=1-10; razred 2).

Growth and photosynthesis of Lemna minor L. exposed to different light conditions and sucrose supplies

Abstract Duckweed (Lemna minor L.) is a model plant suitable for investigation into plant physiology, biochemistry and ecotoxicology. Depending on the type of the experiment, duckweed is cultivated on different nutrient media under various chamber conditions. In our work, duckweed was cultivated on Pirson-Seidel’s nutrient solution supplemented with 5, 7.5 or 10 g L-1 sucrose under cool white (CW) or Gro-Lux (GL) light sources. When different light sources and sucrose supplies are compared, a significant stimulative effect of GL light on duckweed grown on 7.5 and 10 g L-1 sucrose was seen to start on day 9. Considering photosynthetic performance the results showed that there were no significant differences in maximum quantum yield of PSII (Fv/Fm) after 7 and 16 days of exposure, regardless of light source and sucrose supply. Effective quantum yield of PSII (ΦPSII) decreased only after 16 days of exposure to 5 g L-1 sucrose under CW light. The higher growth rate and photosynthetic performance in plants exposed to GL light is a consequence of its spectral distribution resembling the action spectrum of photosynthesis. Furthermore, enhanced growth noticed in plants cultivated on higher sucrose contents (7.5 and 10 g L-1) indicated the promotive effect of sucrose in plants grown under low light conditions.

Response of dihaploid tobacco roots to salt stress

Abstract Salinity is a common abiotic factor that affects plant growth and development. Seedlings of tobacco (Nicotiana tabacum L.) F1 hybrid DH10 and three dihaploid lines (207B, 238C and 239K) obtained by diploidization of anther-derived haploids of hybrid DH10 were subjected to 0, 100 and 200 mM NaCl in in vitro conditions for 33 days and the effect on roots was evaluated. In all lines and in the hybrid DH10 exposed to 200 mM NaCl evident root growth inhibition and increased proline content were noticed. However, in some cases lines differed in the activity of antioxidative enzymes, which could account for differences in their salinity tolerance. Increased activity of catalase and peroxidase in roots of line 239K could contribute to the more pronounced salinity tolerance previously reported for shoots of this line.