Zlatko Mihaljević

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.

Assemblage composition and ecological features of aquatic dance flies (Diptera, Empididae) in the Cetina River system, Croatia

Twenty species of aquatic dance flies (Empididae: Clinocerinae and Hemerodromiinae) were recorded from the Cetina River system, Croatia. No previous such research has been done on any Mediterranean river con-cerning aquatic dance flies. Aduit dance flies were sampled at 7 locations with an aspirator and sweep nets between May and September 2005 and in March and April 2006. The most abundant species was Wiedemannia (Wiedeman-nia) bistigma which was present in ali months but only at locations in the lower reach of the Cetina River. Larvae were sampled at 6 sites from different microhabitat types. Altogether 241 quantitative samples were taken at 20 sampling points between August 2004 and August 2005, but dance flies vvere found only in a small number of the samples. The relationships between dance fly assemblage structure and selected environmental variables (water temperature, dissolved oxygen, alkalinity, conductivity and pH) vvere investigated and showed that the most im-portant environmental variable was water temperature. Some species clearly preferred sites where the mean annual water temperature was constant and lower year round, like springs (e.g., Chelifera spp., Wiedemannia (Philolutra) kacanskae, Wiedemannia (Chamaedipsia) ariadne), while other species preferred sites where the mean annual water temperature was higher (e.g., Hemerodromia spp., Wiedemannia (Wiedemannia) tricuspidata, Wiedeman-nia {; ; ; Pseudowiedemannia) lamelatta). Some species did not show any strong preferences for any of the selected parameters (e.g., Wiedemannia (Eucelidia) zetterstedti, Wiedemannia (Wiedemannia) bistigma, Dolichocephala guttata). A significant positive correlation emerged between the amount of prey present in different microhabitat types and the number of dance fly larvae. A significant difference was also found between microhabitat types and total number of dance fly larvae.

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.

Copepod communities in karstic mediterranean lakes along the eastern Adriatic coast

The copepod communities of karstic lakes along the eastern Adriatic coast were studied. Lakes were divided in several groups according to their morphology (deep, shallow, barrage and reservoirs), production (oligotrophic, mesotophic and eutrophic), and salinity of water: freshwater and brackish. Copidodiaptomus steueri, Eucyclops serrulatusand Macrocyclops albidus belong to the group that inhabited most of the lakes under the study, regardless of lake type. The shallow karstic lakes are usually inhabited by Thermocyclops dybowskii, T. oithonoides, Cyclops vicinus and Eudiaptomus padanus etruscus in freshwater biotopes, and Calanipedia aquaedulcis and Copidodiaptomus steueri in brackish biotopes. The last two species can also be found in deep karstic lakes with brackish water (Bacina lakes in the Neretva River delta). Species like Cyclops abyssorumcan be found in most deep freshwater lakes. Some Calanoida were recorded in only one lake, like Eudiaptomus transsylvanicusin the deep Lake Vrana on the island of Cres, or Eudiaptomus hadzici in the barrage Lake Visovac. Production of the lakes, expressed as copepod biomass, depends on lake trophy, and in some lakes also on hydrology and salinity. Most of the meso-eutrophic lakes in the study area had Calanoida dominating, while Cyclopoida dominated in some oligotrophic and eutrophic lakes as well.

Distribution and Autecology of Mayflies (Insecta, Ephemeroptera) in a Mediterranean River in the Western Balkans

ABSTRACT: Mayflies are one of the most important orders of freshwater inhabiting insects and they are widely used in ecological studies. Mayfly ecology and distribution in the Mediterranean part of the Western Balkans are insufficiently investigated. Our study encompassed 9 sampling sites along the course of the Cetina River, the longest Mediterrranean river in Croatia, and 2 sites in the spring reach of its tributary, Ruda River. Mayflies were sampled monthly between August 2004 and August 2005. A total of 22 mayfly taxa were recorded, including 12 rare species. Due to the variety of available microhabitats and suitable physico-chemical water properties (e.g. water temperature), the highest mayfly richness was recorded in the lower reaches of the river. We discuss distribution and ecology of collected species, particularly the most abundant species (e.g. Baetis rhodani (Pictet, 1843) and Serratella ignita (Poda, 1761)) and the rare species (e.g. Siphlonurus croaticus Ulmer, 1920). One of the rare species collected, Caenis beskidensis Sowa, 1973, has a central and western European distribution and was hitherto not recorded in the Dinaric Western Balkan ecoregion. Morphologically variable specimens from the Rhithrogena group semicolorata were collected, and variability of the most important morphological characters is presented here. In order to resolve the taxonomical status we sequenced the mitochondrial cytochrome oxidase c subunit I (barcode region) of morphologically distinct specimens from the Cetina spring and another locality belonging to the Black Sea drainage (Bijela rijeka, Plitvice Lakes NP). Uncorrected p-distances within and between populations enabled us to associate all specimens to Rhithrogena braaschi Jacob, 1974. Moreover, in contrast to high morphological variability observed in this species, we infer low genetic differentiation of the mtCOI fragment in this species in the Dinaric Western Balkans.

Croatian mayflies (Insecta, Ephemeroptera): species diversity and distribution patterns

Abstract Knowledge of the mayfly biodiversity in the Balkan Peninsula is still far from complete. Compared to the neighbouring countries, the mayfly fauna in Croatia is very poorly known. Situated at the crossroads of central and Mediterranean Europe and the Balkan Peninsula, Croatia is divided into two ecoregions: Dinaric western Balkan and Pannonian lowland. Mayflies were sampled between 2003 and 2013 at 171 sites, and a total of 66 species was recorded. Combined with the literature data, the Croatian mayfly fauna reached a total of 79 taxa. Of these, 29 species were recorded for the first time in Croatia while 15 species were not previously recorded in Dinaric western Balkan ecoregion. Based on the mayfly assemblage, sampling sites were first structured by ecoregion and then by habitat type. In comparison with the surrounding countries, the Croatian mayfly fauna is the most similar to the Hungarian and Bosnian fauna. Some morphologically interesting taxa such as Baetis cf. nubecularis Eaton, 1898 and Rhithrogena from the diaphana group were recorded. Ephemera cf. parnassiana Demoulin, 1958, the species previously recorded only from Greece, was also recorded.

Colonisation trends, community and trophic structure of chironomid larvae (Chironomidae: Diptera) in a temporal phytophylous assemblage

Spatial and temporal trends of a phytophylous chironomid community on the submerged plant species Ceratophyllum demersum L. were investigated in a eutrophic lake (Lake Sakadas, Danube floodplain area in Croatia) during summer 2004. The study also included an analysis of functional feeding groups (FFG) and the influence of environmental parameters on the chironomid community. Within macrophyte stands at three sites, the chironomids were very abundant and the recorded larvae belonged to three subfamilies: Chironominae (Chironomini and Tanytarsini), Orthocladiinae and Tanypodinae. Endochironomus albipennis type was the dominant species with high abundance in July and September and lower abundance in August, most likely due to summer emergence. At the beginning of the sampling season, high abundance was recorded for Cricotopus sylvestris type as well. The secondary colonizers, Polypedilum sordens type, Glyptotendipens pallens type and Parachironomus varus type, displayed an increase in abundance towards the end of the season. ANOSIM analysis indicated significant differences in chironomid abundance between samples from July and September as a result of the higher abundance of Chironomini larvae in September. Active filterers, detritovores and predators were more abundant in September as compared to the other two months, while grazer abundance decreased towards the end of the study period. ANOSIM analysis separated August and September based on the abundance of different functional feeding groups (FFG). We found no significant differences between research stations for either chironomid abundance or FFG. BIO-ENV analysis indicated an influence of environmental variables, especially water temperature, Secchi depth and oxygen concentration on chironomid abundance.