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Chironomids are found in all types of freshwater habitats; they are a ubiquitous and highly diverse group of aquatic insects. Plitvice Lakes National Park is the oldest and largest national park in Croatia and consists of numerous and diverse freshwater habitats, making the area an ideal location for long-term research into the chironomid emergence patterns and phenology. The main objectives of this study were to identify the composition of the chironomid community, determine the phenology of the identified species, and assess the main factors influencing their emergence in Plitvice Lakes. During 14 years of research, more than 13,000 chironomids belonging to more than 80 species were recorded. The most abundant species was found to be Parametriocnemus stylatus . The highest abundance of chironomids was recorded in lotic habitats with faster water current over substrates of moss and algae and pebbles. Water temperature and the availability of organic matter were found to be the main factors that drive chironomid emergence at the tufa barrier studied. In the last years of this study, a prolonged flight period was observed. Although this is not statistically significant (at this stage of the study), it could be due to a higher water temperature in winter.

Global climate change is expected to exacerbate extreme discharge events in freshwater ecosystems as a consequence of changes in precipitation volume and snow cover duration. Chironomid midges were used as a model organism in this study because of their small size and short life cycles, which enable fast colonization of new habitats and great resilience. This resilience is often expressed in easy recolonization after an extreme event. Chironomid samples together with physico-chemical water measurements were collected for 14 years, between 2007 and 2020, in a karst tufa barrier that is part of the Plitvice Lakes National Park in Croatia. More than 13,000 individuals belonging to >90 taxa were collected. Mean annual water temperature increased by 0.1 °C during this period. Multiple change-point analysis revealed three main periods by discharge patterns: the first one from January 2007 to June 2010, the second from July 2010 to March 2013, characterised by extreme low discharge, and the third from April 2013 to December 2020, characterised by an increase in extreme peak discharge values. Based on multilevel pattern analysis, indicator species of the first and the third discharge period were detected. The ecological preferences of these species indicate an environmental change related to the changes in discharge. Along with species composition, functional composition has changed with the abundance of passive filtrators, shredders and predators increasing over time. Species richness and abundance did not change over the period of observation, thus emphasizing the importance of species-level identification in detecting the earliest community response to change that would otherwise be overlooked., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Historically, rivers have been anthropogenically modified for different purposes worldwide (e.g., flood control, drinking water abstraction, and land drainage). Although the majority of European lowland rivers are under the impact of multiple stressors (water quality, hydromorphological alterations, land-use), the consequences of these influences on aquatic macroinvertebrates, including mayflies, have not been sufficiently studied. Therefore, with the aim of providing additional data on the response of mayflies to anthropogenic disturbances in riverine habitats, we studied mayfly assemblages and their relationship to environmental factors along a mid-sized lowland river in Croatia. No significant differences in mayfly species richness and their functional traits were observed among the three habitat groups with different levels of hydromorphological alterations (near-natural, moderately altered, and severely altered habitats). This could be related to the river's connection to the numerous tributaries, and the variety of available microhabitats along the studied system, despite the existing hydromorphological pressures. A stronger relationship was found between mayflies and the physico-chemical water characteristics and land-use. Water temperature, water velocity, oxygen content, and nutrient (e.g., nitrogen, phosphorous) concentrations related to agricultural and urban land-use were found to be the most important factors shaping mayfly assemblages in the studied lotic lowland system. These results can contribute to the planning of management and conservation activities for lowland rivers and their biota according to the requirements of the European Water Framework Directive.

Freshwater ecosystems are endangered worldwide by various human pressures, resulting in dramatic habitat and species loss. Many aquatic invertebrates respond to disturbances in their habitat, and mayflies are among the most sensitive ones. Therefore, we investigated mayfly response to anthropogenic disturbances at 46 study sites encompassing slightly to heavily modified small and mid-sized lowland streams and rivers. Mayfly nymphs were sampled between April and September 2016 using a benthos hand net. A total of 21 species was recorded, with Cloeon dipterum (Linnaeus, 1761) being the most frequently recorded one. Nevertheless, the taxa richness was rather low per site, i.e., between zero and nine. Assemblage structure had a high share of lower reaches and lentic (potamic and littoral) elements, and detritivores (gatherers/collectors and active filter feeders). This indicates that hydromorphological alterations lead to assemblage "potamisation" in small and mid-sized rivers. More mayfly species were related to higher oxygen concentration and lower water temperature, abundance of aquatic vegetation and total organic carbon. Additionally, the assemblage diversity and abundance were negatively associated with increasing intensive agriculture area at the catchment scale. This study confirms mayfly bio-indicative properties, i.e., their sensitivity to alterations of their habitat and pollution, but also provides new data related to mayfly response to the impacted environment. Those data can be used for management and protection activities of lowland rivers and their biota according to the requirements of the European Water Framework Directive., (Marina Vilenica, Mladen Kerovec, Ivana Pozojević, Zlatko Mihaljević.)

The family of non-biting midges (Diptera, Chironomidae) represent one of the most common and abundant group in freshwaters, inhabiting a wide variety of habitats and ecological niches. Although there is a long tradition of limnological research in Croatia, to date no extensive species list has been made for this insect family. Thus, we summarized the results of our research and reviewed published data on chironomid fauna of Croatia in various freshwater habitat types, including extreme ones, such as caves, peatbogs or marine littoral. A total of 239 species were recorded representing five subfamilies: Chironominae (125 species) Orthocladiinae (83 species), Tanypodinae (23 species), Diamesinae (6 species) and Prodiamesinae (2 species). The most frequent species were Cricotopus bicinctus, Dicrotendipes nervosus, Synorthocladius semivirens, each found in more than 20% of the studied localities. Ablabesmyia monilis and Procladius choreus represented the most frequent Tanypodinae species, recorded in more than 12% and 17% of the sites, respectively. The research of chironomid ecology, diversity and distribution continues, which will provide new data and information, but this first comprehensive checklist provides a good starting point for those studying Chironomidae or other freshwater Diptera, in Croatia and Europe.

Studies on aquatic Diptera in the Plitvice Lakes National Park (Croatia) conducted in the last 50 years have produced 157 species and 7 taxa of aquatic Diptera placed in 13 families. Samples were collected at 25 sampling sites representing the four main types of karst aquatic habitats: spring, stream, tufa barriers and lakes. All records of all the aquatic families of Diptera in Plitvice Lakes NP are summarized, including previously unpublished data. Twelve species new for Plitvice Lakes NP are recorded for the first time, belonging to the families: Chironomidae - Labrundinia longipalpis (Goetghebuer, 1921), Nilothauma brayi (Goetghebuer, 1921), Potthastia longimanus Kieffer, 1922, Polypedilum (Polypedilum) nubeculosum (Meigen, 1804), Tanytarsus brundini Lindeberg, 1963; Dixidae - Dixella autumnalis (Meigen, 1838); Scathophagidae - Acanthocnema latipennis Becker, 1894 and Stratiomyidae - Oxycera pardalina Meigen, 1822, Oxycera limbata Loew, 1862, Oxycera turcica Ustuner & Hasbenli, 2004, Nemotelus pantherinus (Linnaeus, 1758), Oplodontha viridula (Fabricius, 1775). The most species-rich family was the Chironomidae with 62 species (and an additional seven taxa), followed by the Empididae with 22 species and Limoniidae with 19 species. The highest number of species was recorded in springs. The relatively low number of species in certain families and the complete absence of some aquatic families shows that further research into the aquatic Diptera in Plitvice Lakes NP is needed., (Marija Ivković, Valentina Dorić, Viktor Baranov, Zlatko Mihaljević, Levente-Péter Kolcsár, Gunnar Mikalsen Kvifte, Jana Nerudova, Adrian C. Pont.)

Predation is a biotic interaction that links water mites to different taxonomic groups of benthic invertebrates. Diptera larvae (Chironomidae) and microcrustaceans (Cladocera, Copepoda, Ostracoda) are known to be the most commonly preferred prey by water mites. Although these predatory interactions are known from observations and ex situ investigations, the distribution patterns and co-occurrence of water mites and their prey in littoral lentic habitats have been poorly studied. Our goal was to determine whether predation might serve as a significant factor in water mite assemblage composition and distribution. Samples were taken from littoral zones of 21 artificial lakes (reservoirs) in the Dinaric Western Balkan ecoregion of Croatia. At every site, 10 samples were collected with regard to shore slope in depth zones of up to 1 m. In total 490 samples were collected from April 2016 to July 2017. Data analysis showed that the spatial distribution of water mites was partially correlated with the environmental parameters we measured (explaining 45.1% of total water mite variation). A positive correlation between water mite abundance and diversity and depth gradient (favouring more stable conditions in deeper parts of the littoral zone) was also observed. Finally, water mite abundance and species richness variation were found to be best predicted by the abundance of potential prey groups. Predation was statistically determined to be a strong dispersion variable, most probably influencing both spatial distribution and composition of water mite assemblages.

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.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (© 2013 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

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.

The diversity, species composition and structure of the fish community were studied in the vicinity of three power plant reservoirs in the middle part of the Drava River in Croatia. Over a period of twenty years in a collection of 25,913 fishes, 56 different fish species belonging to 16 families were recorded. The family Cobitidae is represented by two species: Cobitis elongatoides and Misgurnus fossilis, while the family Balitoridae is represented by only one species, Barbatula barbatula. The presence of the Cobitidae family was recorded in eleven of seventeen investigated localities, while Balitoridae was found in six localities. In the studied fish community, the percentage of C. elongatoides is 1.41% of the total number of individuals, and B. harbatula is 1.02%. M. fossilis is a rare species (0.03 %). Both C. elongatoides and B. barbatula were not caught at the beginning of investigation. C. elongatoides and B. harhatula were first caught in 1985, while M. fossilis was first caught in 1996. The proportion of C. elongatoides in the entire community increased, while the proportion of B. barhatula decreased in later years.