Global warming scenarios for the Eastern Adriatic Sea indicate a higher risk of invasiveness of non-native marine organisms relative to current climate conditions.

Globally, marine bioinvasions threaten marine ecosystem structure and function, with the Mediterranean Sea being one of the most affected regions. Such invasions are expected to increase due to climate change. We conducted a risk screening of marine organisms (37 fishes, 38 invertebrates, and 9 plants), both extant and 'horizon' (i.e., not present in the area but likely to enter it). Based on expert knowledge for the Eastern Adriatic Sea coasts of Slovenia, Croatia, and Montenegro, screenings were conducted under both current and predicted climate conditions indicating with an increase in sea surface temperature and salinity of the Adriatic Sea together with changes in precipitation regime. Our aims were to: (1) identify non-native extant and horizon marine species that may pose threats to native biodiversity and (2) evaluate the risk of invasiveness of the selected species under current and predicted climate conditions. Of the 84 species screened, there was an increase in those ranked as 'high risk' from 33 (39.3%) under current climate conditions and to 47 (56.0%) under global warming scenarios. For those ranked as 'very high' risk, the increase was from 6 (7.1%) to 21 (25.0%). Amongst the screened species, the already established high-risk species Pacific oyster Magallana gigas and Atlantic blue crab Callinectes sapidus represent a threat to ecosystem services. Given the under-representation of marine species in the current European Union List, the species we have ranked as high to very high risk should be included. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00196-9.

In situ detection of the genotoxic potential as one of the lines of evidence in the weight-of-evidence approach-the Joint Danube Survey 4 Case Study.

Environmental studies which aim to assess the ecological impact of chemical and other types of pollution should employ a complex weight-of-evidence approach with multiple lines of evidence (LoEs). This study focused on in situ genotoxicological methods such as the comet and micronucleus assays and randomly amplified polymorphic DNA analysis as one of the multiple LoEs (LoE3) on the fish species Alburnus alburnus (bleak) as a bioindicator. The study was carried out within the Joint Danube Survey 4 (JDS4) at nine sites in the Danube River Basin in the Republic of Serbia. Out of nine sampling sites, two were situated at the Tisa, Sava, and Velika Morava rivers, and three sites were at the Danube River. The three additionally employed LoEs were: SumTUwater calculated based on the monitoring data in the database of the Serbian Environmental Protection Agency (SEPA) (LoE1); in vitro analyses of JDS4 water extracts employing genotoxicological methods (LoE2); assessment of the ecological status/potential by SEPA and indication of the ecological status for the sites performed within the JDS4 (LoE4). The analyzed biomarker responses in the bleak were integrated into the unique integrated biomarker response index which was used to rank the sites. The highest pollution pressure was recorded at JDS4 39 and JDS4 36, while the lowest was at JDS4 35. The impact of pollution was confirmed at three sites, JDS4 33, 40, and 41, by all four LoEs. At other sampling sites, a difference was observed regarding the pollution depending on the employed LoEs. This indicates the importance of implementing a comprehensive weight-of-evidence approach to ensure the impact of pollution is not overlooked when using only one LoE as is often the case in environmental studies.

Long-term analysis of fish assemblage structure in the middle section of the Sava River - The impact of pollution, flood protection and dam construction.

At the beginning of the 20th century, the middle section of the Sava River in Croatia was unaffected by major human activities and rich in ichthyofauna. The Sava River was important for commercial and recreational fishing for the local population, which still remains today. However, the 1920s mining industry was established in Slovenia, which emitted carbon dust into the Sava River. At the same time, the construction of embankments to mitigate flooding started in the middle section. Furthermore, in the 1980s, the Krsko nuclear power plant (NPP), and in the 2010s, the Krsko hydropower plant (HPP) were built in Slovenia. These activities could have an impact on the composition of fish communities downstream from the major sources of disturbances. Therefore, the main aim of this paper were to analyze the changes in fish assemblages of the Sava River from 1978 to 2017, prior to and after the construction of Krsko NPP and HPP at the Medsave site on the Sava River, 20 km downstream from the major construction operations. Collected data were divided into four sampling periods (SP): SP1, from 1978 to 1980; SP2, from 1991 to 1994; SP3, from 2001 to 2006, and SP4 from 2011 to 2017. Besides alien fish species, water quality and hydromorphological modifications were identified as significant stressors. In SP1 and SP2 limnophilic and eurytopic fish groups were predominant, and 26 different fish species were identified, but in SP3 and SP4 rheophilic fish groups become dominant, and the diversity has declined to 21 species. Threatened species blageon, Telestes souffia seems to be missing from the main course of the Sava River in last 20 years. It can be concluded that disturbances in the fish assemblage pattern have coincided with the presence of multiple stressors of human origin.

Potentially toxic elements in muscle tissue of different fish species from the Sava River and risk assessment for consumers.

Fish from the Sava River are consumed daily by the local people: therefore, concern has been raised about the health implications of eating contaminated fish. In the present study, potentially toxic elements (PTE), such as Zn, Cu, Cr, Ni, Cd, Pb, As, Hg, and methylmercury (MeHg), were determined in ichthyofauna that are commonly consumed. PTE were determined in the fish muscle tissue. Fish were sampled at 12 locations from the source of the Sava River to its confluence with the Danube River during two sampling campaigns, namely; in 2014 under high water conditions and in 2015 under normal water conditions. Due to the different water regimes, different fish species were collected for chemical analysis. We observed that the concentrations of elements analysed in the fish muscle tissue were generally very low, except for those of Hg. Moreover, more than 90% of Hg present in the fish was in its most toxic form, namely MeHg. Especially in fish from the 2015 sampling campaign, Hg and MeHg concentrations increased with fish size, trophic level, and in the downstream direction. In addition, for Pb and As, and to some extent for Cd and Cr, spatial differences were detected in both years. The highest concentrations of PTE were detected in fish from sites with intensive industrial and agricultural activities. The consumption of fish in general does not pose a health risk for the PTE studied, except for Hg/MeHg at selected contaminated sites.

Characterization of sections of the Sava River based on fish community structure.

Sampling was undertaken, with the same fishing gear and along the Sava River, from its source to its confluence, in September 2014 and September 2015. In total, 44 fish species were identified, of which 37 were native species and 7 were alien. Fish samples revealed independence in terms of both species composition and their abundance under different hydrological conditions. During flooding and high water levels in 2014, pelagic fish species were sampled in greater proportion than at lower water levels in 2015 when benthic fish species were more abundant. The flood wave in 2014 was accompanied by catch of common carp, Cyprinus carpio, a typical lower rhithron fish species in the upper course, and of tench, Tinca tinca, a typical potamon fish species of backwaters, in the main channel of the lower Sava River. One specimen of bighead goby, Ponticola kessleri, which is common in the potamon fish community, was caught during the 2015 sampling close to the boundary between the upper and middle sections of the Sava. This is the first record of Ponto-Caspian gobies in the inland waters of Slovenia. Its finding far upstream indicates a strong effect of an as yet unidentified stress along the Sava River up to the spot where the bighead goby was sampled. Finally, these results indicate that pelagic fish species are more resistant to the stressful effect of flooding than benthic species, and that the structure of fish communities is influenced/affected by flooding as a short-term stressor. The progressively increasing number of alien fish species downstream in the Sava River point to the effects of long-term human-induced stressors in the area.

Evaluation of Genotoxic Pressure along the Sava River.

In this study we have performed a comprehensive genotoxicological survey along the 900 rkm of the Sava River. In total, 12 sites were chosen in compliance with the goals of GLOBAQUA project dealing with the effects of multiple stressors on biodiversity and functioning of aquatic ecosystems. The genotoxic potential was assessed using a complex battery of bioassays performed in prokaryotes and aquatic eukaryotes (freshwater fish). Battery comprised evaluation of mutagenicity by SOS/umuC test in Salmonella typhimurium TA1535/pSK1002. The level of DNA damage as a biomarker of exposure (comet assay) and biomarker of effect (micronucleus assay) and the level of oxidative stress as well (Fpg-modified comet assay) was studied in blood cells of bleak and spirlin (Alburnus alburnus/Alburnoides bipunctatus respectively). Result indicated differential sensitivity of applied bioassays in detection of genotoxic pressure. The standard and Fpg-modified comet assay showed higher potential in differentiation of the sites based on genotoxic potential in comparison with micronucleus assay and SOS/umuC test. Our data represent snapshot of the current status of the river which indicates the presence of genotoxic potential along the river which can be traced to the deterioration of quality of the Sava River by communal and industrial wastewaters. The major highlight of the study is that we have provided complex set of data obtained from a single source (homogeneity of analyses for all samples).

Evaluation of genotoxic potential throughout the upper and middle stretches of Adige river basin.

In this study a comprehensive genotoxicological survey throughout the upper and middle stretches of Adige river basin is presented. The study was carried out at 7 sites located along the Adige main course and one the most significant tributaries, the Noce creek, both presenting different levels of pollution pressure. To give an insight into the nature of the genotoxic activity we employed the battery of prokaryotic and eukaryotic assays. Mutagenicity in water samples was evaluated by SOS/umuC test in Salmonella typhimurium TA1535/pSK1002. The level of DNA damage as a biomarker of exposure (comet assay) and biomarker of effect (micronucleus assay) and the level of oxidative stress as well (Fpg - modified comet assay) were studied in blood cells of Salmo cenerinus Nardo, 1847 and Salmo marmoratus Cuvier, 1829. Within the applied bioassays, comet assay showed the highest potential for discriminating the sampling sites which are under lesser extent of pressure (sampling sites 1-Barnes at Bresimo and 4-Noce downstream S. Giustina) from the sites under high pressure (sampling sites 5-Noce at Mezzolombardo and 6/7-Adige upstream and downstream municipality of Trento). Significant correlation between the standard and Fpg - modified comet assay indicated that oxidative stress could be a major contributor to observed DNA damage in collected specimens.

Biochemical indicators and biomarkers in chub (Squalius cephalus L.) from the Sava River.

Biochemical indicators and biomarkers were analyzed in the liver and gills of chub caught in three localities along the Sava River exposed to different environmental impacts. Sampling sites were: downstream from Zagreb (Zgd), downstream Sremska Mitrovica (SM) and downstream from Belgrade (Bgd). We observed that the relative amounts and levels of activity of Cu, Zn containing superoxide dismutase and glutathione in both the liver and gills, and the relative amounts of heat shock protein (HSP90) and metallothioneins in the gills were highest in the Zgd locality, suggesting a higher impact of metal pollution. The Zgd locality had higher concentrations of trace metals in the water, especially iron. In the SM and Bgd localities, higher relative levels of glutathione peroxidase and catalase were recorded (especially in SM) as compared to the Zgd locality, pointing to the presence of hydrogen peroxide and different classes of organic peroxides. Low water oxygen and high temperature levels in the Bgd locality suggesting different metabolic activity between examined locations. Our results suggest that different presence and concentrations of individual environmental factors (total environment) influence the way how fish establish homeostasis.

Bioaccumulation of Heavy Metals and Microelements in Silver Bream (Brama brama L.), Northern Pike (Esox lucius L.), Sterlet (Acipenser ruthenus L.), and Common Carp (Cyprinus carpio L.) From Tisza River, Serbia.

The aim of this study was to determine the concentrations of Al, As, B, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Sr, and Zn in liver, gills, gonads, and brain of four ecologically different fish species in Serbia: piscivorous northern pike, benthivorous sterlet and silver bream, and omnivorous common carp. Fish were caught at four sites along the stretch of the River Tisza in the Pannonian part of Serbia during October 2010. Results revealed that heavy metals and microelements with the highest values in fish samples were Fe, Al, and Zn. The highest concentration of heavy metals and microelements was recorded in omnivorous common carp, and organs that most intensively accumulated the greatest number of metals were liver and gills, whereas the locality did not exert a marked impact on level of bioaccumulation.

Socio-economic features of commercial fishery in the bordering upper Danube River area of Serbia.

The multidisciplinary socio-economic study of fisheries in the bordering part of the Danube River between Serbia and Croatia (at the following sites: Apatin, Backa Palanka, Backo Novo Selo, Bezdan, and Sombor) that was performed in order to investigate various aspects of fish resource utilization (management, policy of protection and exploitation of freshwater fishery resources, present fisheries legislation, catch statistics), was realized during 2004 and 2005. Data were collected via survey with a structured interview. Socio-economic circumstances, together with ecological factors, have had an influence on the fish stock and number of commercial fishermen. Awareness of the occurring problems, both economic and ecological ones, is apparent, regardless of whether it is assessed in the field of commercial or recreational fishing. Fishery sector in Serbia is in a prolonged process of transition, with the enforcement of fishing regulations, but also the lack of control that leaves space for illegal commercial fishing. The statements, consciousness, experience and behavior of commercial fishermen represent a good basis for planning the sustainable development of fishing in this section of the Danube River.

Genetic differentiation of European grayling (Thymallus thymallus) populations in Serbia, based on mitochondrial and nuclear DNA analyses.

BACKGROUND: The structure and diversity of grayling (Thymallus thymallus) populations have been well studied in most of its native habitat; however the southernmost populations of the Balkan Peninsula remain largely unexplored. The purpose of this study was to assess the genetic diversity of Serbian grayling populations, detect the impact of stocking and provide guidelines for conservation and management. METHODS: Eighty grayling individuals were collected from four rivers (Ibar, Lim, Drina and Rzav). The mitochondrial DNA control region (CR; 595 bp of the 3'end and 74 bp of flanking tRNA) and the ATP6 gene (630 bp fragment) were sequenced for 20 individuals (five from each locality). In addition, all individuals were genotyped with 12 microsatellite loci. The diversity and structure of the populations as well as the recent and ancient population declines were studied using specialized software. RESULTS: We detected three new haplotypes in the mtDNA CR and four haplotypes in the ATP6 gene of which three had not been described before. Previously, one CR haplotype and two ATP6 gene haplotypes had been identified as allochthonous, originating from Slovenia. Reconstruction of phylogenetic relations placed the remaining two CR haplotypes from the River Danube drainage of Serbia into a new clade, which is related to the previously described sister Slovenian clade. These two clades form a new Balkan clade. Microsatellite marker analysis showed that all four populations are genetically distinct from each other without any sign of intra-population structure, although stocking of the most diverse population (Drina River) was confirmed by mtDNA analysis. Recent and historical population declines of Serbian grayling do not differ from those of other European populations. CONCLUSIONS: Our study shows that (1) the Ibar, Lim and Drina Rivers grayling populations are genetically distinct from populations outside of Serbia and thus should be managed as native populations in spite of some introgression in the Drina River population and (2) the Rzav River population is not appropriate for further stocking activities since it originates from stocked Slovenian grayling. However, the Rzav River population does not represent an immediate danger to other populations because it is physically isolated from these.

Around or across the Carpathians: colonization model of the Danube basin inferred from genetic diversification of stone loach (Barbatula barbatula) populations.

Despite increasing information about postglacial recolonization of European freshwater systems, very little is known about pre-Pleistocene history. We used data on the recent distribution and phylogenetic relationships of stone loach mitochondrial lineages to reconstruct the initial colonization pattern of the Danube river system, one of the most important refuges for European freshwater ichthyofauna. Fine-scale phylogeography of the Danubian populations revealed five highly divergent lineages of pre-Pleistocene age and suggested the multiple origin of the Danubian stone loach. The mean sequence divergence among lineages extended from 7.0% to 13.4%, which is the highest intraspecific divergence observed so far within this river system. Based on the phylogeographical patterns, we propose the following hypothesis to relate the evolution and dispersal of the studied species with the evolution of the Danube river system and the Carpathian Mountains: (i) during the warmer period in the Miocene, the areas surrounding the uplifting Alps and Carpathians served as mountainous refuges for cold-water adapted fish and promoted the diversification of its populations, and (ii) from these refuges, colonization of the emerging Danube river system may have taken place following the retreat of the Central Paratethys. Co-existence of highly divergent mtDNA lineages in a single river system shows that range shifts in response to climatic changes during the Quaternary did not cause extensive genetic homogenization in the stone loach populations. However, the wide distribution of some mtDNA lineages indicates that the Pleistocene glaciations promoted the dispersal and mixing of populations through the lowlands.

Phylogeographic study of brown trout from Serbia, based on mitochondrial DNA control region analysis.

In order to illuminate the phylogeography of brown trout (Salmo trutta) populations in the Balkan state of Serbia, the 561 bp 5'-end of mtDNA control region of 101 individuals originating from upland tributaries of the Danubian, Aegean and Adriatic drainages were sequenced and compared to corresponding brown trout sequences obtained in previous studies. Among 15 haplotypes found, 14 were considered native, representing the Danubian and Adriatic lineages of the brown trout, while one haplotype (ATcs1), found only in two individuals originating from two stocked rivers, corresponded to the Atlantic lineage and was considered introduced. Native haplotypes exhibited a strong geographic pattern of distribution: the Danubian haplotypes were strictly confined to the Danubian drainage, while the Adriatic haplotypes dominated in the Aegean and Adriatic drainages; most of the total molecular variance (69%) was attributed to differences among the drainages. Phylogenetic reconstruction, supplemented with seven haplotypes newly described in this study, suggested a sister position of the Atlantic-Danubian and Adriatic-Mediterranean-marmoratus ("southern") phylogenetic group, and pointed to the existence of a distinct clade, detected within the "southern" group. The data obtained confirmed our expectation of the existence of high genetic diversity in Balkan trout populations, and we recommend more widespread surveys covering trout stocks from the region.