Ivančica Ternjej

Seasonal distribution and morphological changes of Ceratium hirundinella in two mediterranean shallow lakes

Seasonal distribution and morphological changes in two forms of Ceratium hirundinella in the shallow karstic Mediterranean lakes, Ponikve and Njivice, during 1997/98 and 2000/01, were studied. C. hirundinella f. gracile was present during the whole investigation period in Lake Njivice. The same species was the dominant form in Ponikve Lake during 1997/98, while in 2000/01 a strong development of C. hirundinella f. austriacum occurred. The species C. hirundinella f. austriacum was restricted to spring–summer time. This population showed larger variations in number of horns. An analysis of morphometric measurements showed significant differences (p<0.01) in the total length of cells, length of the fourth horn and V3 angle of the two Ceratium forms.

Estimation of DNA Integrity in Blood Cells of Eastern Mosquitofish (Gambusia holbrooki) Inhabiting an Aluminium-Polluted Water Environment: an Alkaline Comet Assay Study

To estimate the impacts of an Al-contaminated aquatic environment on DNA integrity in the blood cells of eastern mosquitofish Gambusia holbrooki Girard 1859 inhabiting Lake Njivice (Island of Krk, Croatia), an evaluation using the alkaline comet assay was carried out. Genome integrity was studied in parallel with the same fish species inhabiting the nearby, unpolluted Lake Ponikve. The amount of DNA damage in cells was estimated from three different parameters: comet tail length as the extent of genetic material migration, tail intensity (% DNA in the comet tail) and tail moment. The results indicate the loss of genome integrity in blood cells of mosquitofish inhabiting Lake Njivice and the genotoxicity of this aquatic environment. Using the same assay, acute genotoxicity of contaminated water and sediment was evaluated and confirmed on fish, mouse and human blood cells treated ex vivo. Results of the present study indicate that the alkaline comet assay applied to fish blood cells is a valuable tool for determining the potential genotoxicity of water pollutants and confirm its usefulness in the evaluation of DNA damage in fish living in Al-polluted waters.

Application of the comet assay and detection of DNA damage in haemocytes of medicinal leech affected by aluminium pollution: A case study

This report describes an investigation of genotoxic effects in medicinal leech (Hirudo verbana) exposed to water and sediment of Lake Njivice (Krk Island, Croatia) contaminated by aluminium compounds. The levels of primary DNA damage in leech haemocytes and loss of DNA integrity caused by acute and chronic exposure to contaminated water and sediment were investigated using the alkaline comet assay. Genotoxic effects induced by acute exposure to contaminants were evaluated on leech haemocytes and blood cells of fish and mouse treated ex vivo. The effects of chronic exposure were assessed on haemocytes sampled from an animal kept under laboratory conditions on contaminated water and sediment for 180 days. The results indicate the DNA damaging potential of aluminium compounds present in an excess amount in tested samples.

Crustacean plankton community (Crustacea: Copepoda and Cladocera) in gypsum karst lakes and their relation to abiotic parameters

Two gypsum karst lakes and one non-gypsum karst lake were studied for copepod and cladoceran species composition in relation to specific habitat characteristics. The investigation was conducted from March to June 2007. Four copepod and six cladoceran species were recorded. Gypsum karst lakes are characterized as sulphate lakes and they show significant differences from non-gypsum karst lakes in conductivity, TDS, alkalinity, calcium and sulphates. Data on environmental variables and zooplankton were analyzed using redundancy analysis (RDA). The model explained 65.73% of the variance of the crustacean zooplankton and environmental data by the first two axes. The analysis confirmed that the major environmental variables influencing zooplankton in gypsum karst lakes are conductivity, TDS, calcium and sulphates. In the non-gypsum karst lake, on the contrary, the major variables were oxygen concentration and alkalinity. Specific habitat characteristics of gypsum karst lakes influence the zooplankton community by reducing the number of species and leading to the dominance of one of them.

Assessment of genotoxic potency of sulfate-rich surface waters on medicinal leech and human leukocytes using different versions of the Comet assay.

The aim of the present study was to investigate how exposure to sulfate-rich surface waters affects the level of primary DNA damage in hemocytes of leech Hirudo medicinalis. Samples of surface water were collected at two sites near a gypsum factory (Knin, Croatia) and two reference sites. In the laboratory, samples were subjected to detailed chemical analysis and used in toxicity testing. For that purpose, previously acclimatized individuals of H. medicinalis were sub-chronically exposed (for 28 days) to tested water samples. Levels of primary DNA damage were evaluated using the alkaline Comet assay in hemocytes collected on days 7, 14, 21 and 28 of exposure and compared with their baseline values. Genotoxic potency of the water sample with the highest sulfate concentration was further evaluated using the alkaline, neutral and hOGG1-modified Comet assay on human peripheral blood leukocytes exposed ex vivo for 30 min. The purpose was to explore which mechanisms are responsible for DNA damage. Chemical analysis revealed that sulfate concentrations in two water samples collected in Mali Kukar Lake (1630 mg/L SO₄) and Kosovčica River (823.3 mg/L SO₄) exceeded the WHO and US EPA defined limits for sulfate in drinking water. Increased levels of metals were found only in the water sample collected in Mali Kukar Lake. However, of the 65 elements analyzed, only nickel and titanium exceed the value legally accepted in Croatia for drinking water. The levels of DNA damage, estimated by the alkaline Comet assay in hemocytes of medicinal leech, increased with the duration of exposure to two sulfate-rich water samples. Since hemocytes responded sensitively to treatment, they could be used for biomonitoring purposes. As observed on treated human peripheral blood leukocytes, all versions of the Comet assay were effective in detecting DNA damage, which was measured in samples with sulfate concentrations equal to or higher than the legally accepted levels for drinking water. Based on the obtained results, it can be assumed that genotoxicity was a consequence both of direct (single- and double-strand DNA breaks) and indirect effects (oxidative damage) caused by the combined effects of all contaminants present in the tested water samples. Our results indicate the need for in situ monitoring and purification of gypsum mine water prior to its release in the natural environment.

Cytotoxic and genotoxic effects of water and sediment samples from gypsum mining area in channel catfish ovary (CCO) cells

Man-made activities such as mining generate certain amounts of metal contaminated wastes which can reach aquatic environment and cause the serious effects on different organisms and ecosystem. Chemical analysis of the environmental samples is the most direct approach to reveal their pollution status but it cannot always provide information on biological effects to different organisms, including fish. This study was aimed to investigate the in vitro cytotoxicity and genotoxicity of water and sediment samples from gypsum mining area using the channel catfish ovary (CCO) cell line. Results obtained by the WST-1 assay and alkaline comet assay revealed that exposure of CCO cells to the same concentrations of contaminated water and sediment samples caused significant decrease in cell viability and increased DNA damages. Chemical analysis of water and sediment samples showed that increased concentrations of strontium, aluminum and iron were mainly responsible for the observed cytotoxic and genotoxic effects in CCO cells. The study suggested that fish CCO cells could be useful biological test-system for water and sediment cytotoxicity and genotoxicity assessments.

Cryptic diversity in the Western Balkan endemic copepod: Four species in one?

We use mitochondrial (mtCOI) and nuclear (nH3) sequence data to investigate differentiation of Eudiaptomus hadzici, a freshwater copepod endemic to the Western Balkans. E. hadzici has a disjunct distribution and morphological differences were observed at regional scale. In the current study 6 out of 7 known populations are included. We applied several species delimiting approaches, distance based methods (K2P p-distance and Automatic Barcode Gap Discovery, ABGD) using the mtCOI, Bayesian phylogeny and the Bayesian method implemented in bPTP and BPP programs using the concatenated sequences of both genes. Phylogenetic and species delimitation analyses all suggest that the nominal species E. hadzici consists of four isolated, cryptic evolutionary lineages in the Western Balkans. Each of the four lineages inhabits a single lake or a group of lakes in close proximity. They exhibit major differences in secondary sexual characters, e.g. right antennule in males. Denticulation of spine on 13th segment is substantially distinct among the four lineages, having different number and shape of tooth-like protrusions. Gene flow and dispersal are restricted to very small spatial scale, but with local differences, implying that diverse historical and contemporary processes are operating at small spatial scales in E. hadzici. In order to further examine spatial and temporal diversification patterns, we constructed a dated species tree analysis using (*)BEAST. Due to lack of reliable calibration points and taxa specific evolutionary rates, two evolutionary rates were applied and the faster one (2.6% myr) seems more plausible considering the geological history of the region. The divergence of E. hadzici lineages is dated from Early Miocene onwards with geographically close lineages diverging more recently, Late Miocene to Pleistocene and Pleistocene, respectively. Overall, our findings shed light on cryptic genetic complexity of endemics in one of European biodiversity hotspots. Moreover, this study represents one further example of integrative taxonomy, linking DNA methodology and classical taxonomy based on morphology. Therefore, it lays groundwork for future taxonomy and biogeography of freshwater microcrustaceans in the region.

Taxon specific response of carabids (Coleoptera, Carabidae) and other soil invertebrate taxa on invasive plant Amorpha fruticosa in wetlands

False indigo (Amorpha fruticosa L.) is an invasive exotic plant introduced to Europe in the early eighteenth century. Its spread has been rapid, particularly in disturbed wetland habitats, where it forms dense impermeable monospecific stands and modifies habitat conditions. The impact of A. fruticosa on native plant communities has been well analyzed, however knowledge concerning the possible effects on soil invertebrates and particularly carabid beetles is completely lacking. This study analyzed the impact of an A. fruticosa invasion on carabid beetles and other soil invertebrates. Soil fauna was sampled by pitfall traps at natural habitats, initially colonized by A. fruticosa, and habitats largely invaded by A. fruticosa. In total 2,613 carabid beetles belonging to 50 species and 72,166 soil invertebrates were collected. The invasion of A. fruticosa strongly affected the carabid beetle species composition, which clearly differed between all studied sites. Widespread euritopic carabid beetle species showed positive responses to A. fruticosa invasion, while the activity density of open habitat species strongly declined. Mean individual biomass was significantly higher at invaded sites due to increased incidence of large carabids (genus Carabus Linné, 1758). Carabid beetle activity density and abundance of soil invertebrates were considerably higher at invaded sites than in natural sites. Conversely, the impact of A. fruticosa on carabid beetle species richness and diversity was less pronounced, most likely due to immigration from adjacent habitats. Changes in carabid beetle species composition and abundance of soil invertebrates were most likely due to changes in vegetation structure and microclimate. The results suggest that A. fruticosa invasion considerably affected carabid beetles, an insect group that is only indirectly related to plant composition. Therefore, severe future changes can be expected in invertebrate groups that are closely related to plant composition, since A. fruticosa cannot be completely removed from the habitat and covers relatively large areas.

New ecological insight on two invasive species: Craspedacusta sowerbii (Coelenterata: Limnomedusae) and Dreissenia polymorpha (Bivalvia: Dreissenidae)

The ecology of polyps of Craspedacusta sowerbii has been poorly investigated. It is known that they live attached to hard surfaces. They have never been linked with any animal, in particular not with Dreissena polymorpha (zebra mussel), which is known to have only endosymbionts. This study was conducted in March 2004 in Čingi-Lingi Lake. The lake is an oligo-mesotrophic man-made gravel pit in northeast Croatia. During this study, polyps were found singly or in colonies of two to five on willow twigs and on the shells of D. polymorpha collected from 8 m depth and on metal barrels. Brown hydra (Pelmatohydra oligactis) individuals were also present next to polyps of Craspedacusta on zebra mussel shells, collected from metal barrels. The presence of polyps of Craspedacusta and brown hydras together on live bivalve shells is significant, as we suggest that it marks the first record of commensalism of this kind.

The impact of gypsum mine water: A case study on morphology and DNA integrity in the freshwater invertebrate, Gammarus balcanicus

The aim of our study was to investigate how exposure to heavy metal-rich waters from gypsum mining affects the morphology and levels of primary DNA damage in Gammarus balcanicus. Chemical analysis revealed increased concentrations of metals in water and sediment collected at a site impacted by gypsum mine wastewaters. The specimens also showed elevated total tissue metal levels when compared with the organisms collected at the reference site. The most prominent increase was observed for strontium, followed by iron, nickel, vanadium, aluminium, and manganese. The major pathway of entry for these toxic substances was through the degraded exoskeleton as a consequence of excessive strontium input (unbalanced calcium/strontium ratio) and altered permeability. Disturbed exoskeleton integrity was observed only in individuals collected downstream of the gypsum mine, which was confirmed by electron microscopy. Levels of primary DNA damage were evaluated using the alkaline comet assay in the haemolymph of the specimens.