Pollution impact on metal and biomarker responses in intestinal cytosol of freshwater fish.

In the present study, essential and nonessential metal content and biomarker responses were investigated in the intestine of fish collected from the areas polluted by mining. Our objective was to determine metal and biomarker levels in tissue responsible for dietary intake, which is rarely studied in water pollution research. The study was conducted in the Bregalnica River, reference location, and in the Zletovska and Kriva Rivers (the Republic of North Macedonia), which are directly influenced by the active mines Zletovo and Toranica, respectively. Biological responses were analyzed in Vardar chub (Squalius vardarensis; Karaman, 1928), using for the first time intestinal cytosol as a potentially toxic cell fraction, since metal sensitivity is mostly associated with cytosol. Cytosolic metal levels were higher in fish under the influence of mining (Tl, Li, Cs, Mo, Sr, Cd, Rb, and Cu in the Zletovska River and Cr, Pb, and Se in the Kriva River compared to the Bregalnica River in both seasons). The same trend was evident for total proteins, biomarkers of general stress, and metallothioneins, biomarkers of metal exposure, indicating cellular disturbances in the intestine, the primary site of dietary metal uptake. The association of cytosolic Cu and Cd at all locations pointed to similar pathways and homeostasis of these metallothionein-binding metals. Comparison with other indicator tissues showed that metal concentrations were higher in the intestine of fish from mining-affected areas than in the liver and gills. In general, these results indicated the importance of dietary metal pathways, and cytosolic metal fraction in assessing pollution impacts in freshwater ecosystems.

A preliminary study of the cultivable microbiota on the plastic litter collected by commercial fishing trawlers in the south-eastern Adriatic Sea, with emphasis on Vibrio isolates and their antibiotic resistance.

Mediterranean Sea is the sixth largest area of marine litter accumulation in the world, and plastic pollution is a growing problem in its Adriatic sub-basin. The aim of the present study was to evaluate the cultivable microbiota associated with plastic litter collected by commercial fishing trawlers in the south-eastern Adriatic Sea in comparison with microbiota in seawater and sediment. Plastic litter in the sea contains an autochthonous microbiota that is different from that of the surrounding seawater and sediment. Vibrio abundance was higher on plastic litter than in surrounding seawater and sediment. All isolated Vibrio showing resistance to ampicillin and vancomycin, while resistance to other antibiotics depended on the isolated species. Overall, this study provides for the first time information on the cultivable microbiota associated with plastic litter collected by commercial fishing trawlers and provides a data base for further studies.

Characterization of Vibrio Populations from Cultured European Seabass and the Surrounding Marine Environment with Emphasis on V. anguillarum.

Vibrio species are widely distributed and can be potentially pathogenic to aquatic organisms. In this study, we isolated Vibrio spp. from environmental samples (seawater, sediment, and fish swabs) collected over a three-year period from a fish farm in Mali Ston Bay in the Adriatic Sea, Croatia, and assess their distribution. A total of 48 seawater samples and 12 sediment samples, as well as gill and skin swabs from 110 farmed European seabass, were analysed for the presence of Vibrio. Vibrio strains were identified to the species level by MALDI TOF MS. The analysis revealed that V. alginolyticus was the predominant species in European seabass, followed by V. anguillarum. V. alginolyticus was isolated from the sediments, along with V. gigantis and V. pomeroyi, while V. chagasii, V. cyclitrophicus, V. fortis, V. gigantis, V. harveyi, V. pelagius, and V. pomeroyi were isolated from seawater. V. anguillarum was isolated only twice during two different spring seasons, once from a diseased sea bass and the second time from a healthy sea bass. We analysed these two isolates and found that they differ both genetically and in terms of resistance to antibiotics. Our results confirm the seasonality of vibriosis incidence and the presence of the pathogenic V. anguillarum, which increases the risk of vibriosis.

Microbiological Quality Assessment of Water and Fish from Karst Rivers of the Southeast Black Sea Basin (Croatia), and Antimicrobial Susceptibility of Aeromonas Isolates.

Karst rivers are of great interest for commercial fishing and aquaculture, yet they are quite vulnerable aquatic environments because the permeable karst rocks do not effectively filter out contaminants. To understand the current state of karst rivers water quality, we analysed the physico-chemical and microbiological parameters, focusing on antibiotic pollution and the emergence of antibiotic-resistant microbes of three such rivers in Croatia. Water quality varied between classes I and II across sampling sites, and the numbers of total coliforms, enterococci and heterotrophic bacteria varied substantially among sites. Swabs from fish gills, spleen, liver and kidneys were cultured and 94 isolates identified by MALDI-TOF mass spectrometry. The predominant genus was Aeromonas (42.5% of all identified isolates), known for its adaptability to polluted environments and its frequent association with antibiotic resistance. Of the selected Aeromonas isolates known as most pathogenic, half were resistant to at least three antibiotic categories. The Enterobacteriaceae family was represented by the greatest number of genera, most of which are pathogenic for humans and animals and are spoilage bacteria for fish. The results of this study highlight the extent of antibiotic contamination in aquatic environments and the increasing threat of pathogenic and spoilage bacteria in traditionally high-quality karst rivers.

Influence of technological and municipal wastewaters on vulnerable karst riverine system, Krka River in Croatia.

Exceptional natural value of karst rivers, as well as their potential as a source of drinking water supply, contributes to the importance of developing strict environmental protection. Although most of its watercourse is proclaimed national park, Krka River is impacted by technological and municipal wastewaters, which are released without proper purification only 2 km upstream of the park border. In order to assess water ecological status of the Krka River, vulnerability of karst ecosystem and potential threat to the Krka National Park, spatial and temporal variability of physico-chemical and microbiological water parameters, as well as concentrations of 25 total dissolved metals/metalloids were evaluated. All parameters indicated deteriorated water quality near the wastewater influences, which was for certain metals and bacterial counts even comparable to other world rivers of technological/rural catchment. Downstream location pointed to purification processes in karst river, although nutrients and conductivity were still in a range below good water quality and Al, Co, Fe, Li, Mn, Ni, Sr, Ti, and Zn levels remained comparable to their levels in the pollution impacted area, especially in autumn, indicating season-dependent processes. Presented data provided background status and identified pollution influences in the karst riverine system which indicated potential risk for protected area and a need for comprehensive water quality assessment.

Pomphorhynchus laevis (Acanthocephala) from the Sava River basin: New insights into strain formation, mtDNA-like sequences and dynamics of infection.

Here we report the genetic variability and presence of mtDNA-like sequences of Pomphorhynchus laevis from the chub, Squalius cephalus, caught at the sampling sites along the Sava River and its tributary the Sutla River in Croatia. Sequences of the cytochrome c oxidase subunit 1 (COI) gene of the recovered P. laevis specimens were used for haplotype network construction and phylogenetic analysis. These analyses showed that some specimens contained mitochondrial-like sequences, and they uncovered the existence of a Sava River basin strain different from known strains of P. laevis. This is the first time that P. laevis has been shown to contain mtDNA-like sequences, suggesting the need to exercise caution during COI analyses of P. laevis using universal primers. Highly conserved sequences of two nuclear markers, the ITS region and 18S rRNA, were not helpful for understanding genetic variability or differentiating strains. Furthermore, analysis of the dynamics of P. laevis infections in S. cephalus from the Sava and Sutla Rivers showed decreased prevalence and abundance at sites with inferior water quality, positive association of parasite abundance with fish size, and no clear association of parasite abundance with fish condition index or sex.

Does fish reproduction and metabolic activity influence metal levels in fish intestinal parasites, acanthocephalans, during fish spawning and post-spawning period?

Application of fish intestinal parasites, acanthocephalans, as bioindicators in metal exposure assessment usually involves estimation of their metal levels and bioconcentration factors. Metal levels in parasite final host, fishes, are influenced by fish physiology but there is no data for acanthocephalan metal levels. Gastrointestinal Zn, Fe, Mn, Cd, Ag levels in European chub (Squalius cephalus L.) from the Sava River were significantly higher during chub spawning (April/May) compared to the post-spawning period (September). In acanthocephalans (Pomphorhynchus laevis and Acanthocephalus anguillae) significantly higher metal levels during chub spawning were observed only for Zn in P. laevis. Bioconcentration factors were twice as high for Fe, Mn, Ag, Pb in the post-spawning period, probably as a consequence of lower gastrointestinal metal levels in fish rather than metal exposure. Therefore, bioconcentration factors should be interpreted with caution, due to their possible variability in relation to fish physiology. In addition, gastrointestinal Cu, Cd and Pb levels were lower in infected than uninfected chub, indicating that metal variability in fishes might be affected by the presence of acanthocephalans.

Effect of acanthocephalan infection on metal, total protein and metallothionein concentrations in European chub from a Sava River section with low metal contamination.

In the present study, the importance of considering fish intestinal parasites i.e. the acanthocephalans in metal exposure assessment was estimated under low metal contamination conditions. Two acanthocephalan species, Pomphorhynchus laevis and Acanthocephalus anguillae were examined in 59 specimens of European chub (Squalius cephalus L.) sampled at 5 locations along the Sava River, Croatia. Concentrations of essential (Cu, Mn) and non-essential (Ag, Cd, Pb) metals were higher in intestinal parasites than chub gastrointestinal tissue, but levels of essential metals Fe and Zn were comparable or lower in parasites, respectively. The highest accumulation in both acanthocephalan species was found for non-essential metals and followed the order: Ag>Pb>Cd. Higher infection intensity with P. laevis allowed us to present their spatial metal distribution and evaluate the influence of P. laevis on metal levels and sub-cellular biological responses (total protein and metallothionein levels) in the host infected with P. laevis. Even in the river section with low metal contamination, parasitism affected metal levels, resulting in lower Cu, Cd and Pb concentrations in chub infected with P. laevis than in uninfected chub. Although total protein and metallothionein levels remained constant in infected and uninfected chub, acanthocephalans should be considered a potential confounding factor in metal exposure assessments. Moreover, P. laevis-chub system can be suggested as an appropriate tool in biomonitoring, since in both species increased Cu and Cd concentrations towards the downstream locations were found. Higher Cu and Cd levels in P. laevis suggest acanthocephalans to be sensitive bioindicators if low metal levels have to be detected.

Assessment of general condition of fish inhabiting a moderately contaminated aquatic environment.

The assessment of general condition of fish in the moderately contaminated aquatic environment was performed on the European chub (Squalius cephalus) caught in September 2009 in the Sutla River in Croatia. Although increases of the contaminants in this river (trace and macro elements, bacteria), as well as physico-chemical changes (decreased oxygen saturation, increased conductivity), were still within the environmentally acceptable limits, their concurrent presence in the river water possibly could have induced stress in aquatic organisms. Several biometric parameters, metallothionein (MT), and total cytosolic protein concentrations in chub liver and gills were determined as indicators of chub condition. Microbiological and parasitological analyses were performed with the aim to evaluate chub predisposition for bacterial bioconcentration and parasitic infections. At upstream river sections with decreased oxygen saturation (~50%), decreased Fulton condition indices were observed (FCI: 0.94 g cm(-3)), whereas gonadosomatic (GSI: 2.4%), hepatosomatic (HSI: 1.31%), and gill indices (1.3%) were increased compared to oxygen rich downstream river sections (dissolved oxygen ~90%; FCI: 1.02 g cm(-3); GSI: 0.6%; HIS: ~1.08%; gill index: 1.0%). Slight increase of MT concentrations in both organs at upstream (gills: 1.67 mg g(-1); liver: 1.63 mg g(-1)) compared to downstream sites (gills: 1.56 mg g(-1); liver: 1.23 mg g(-1)), could not be explained by induction caused by increased metal levels in the river water, but presumably by physiological changes caused by general stress due to low oxygen saturation. In addition, at the sampling site characterized by inorganic and fecal contamination, increased incidence of bacterial bioconcentration in internal organs (liver, spleen, kidney) was observed, as well as decrease of intestinal parasitic infections, which is a common finding for metal-contaminated waters. Based on our results, it could be concluded that even moderate contamination of river water by multiple contaminants could result in unfavourable living conditions and cause detectable stress for aquatic organisms.