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U radu su prikazani dužinsko-maseni odnosi i kondicija srdele (Sardina pilchardus) i europskog inćuna (Engraulis encrasicolus) sa područja srednjeg Jadrana (Dugi otok). Tijekom travnja 2021. godine iz ukupnog ulova okružujućom mrežom plivaricom izuzeto je 100 uzoraka srdele i 110 uzoraka inćuna. Dužinsko-maseni odnosi pokazali su pozitivan alometrijski rast obje istraživane vrste (srdela W= 0, 004 x L3, 167, R2=0, 861), (inćun W=0, 002 x L3, 405, R2=0.936). Prosječni Fultonov faktor kondicije analiziranih jedinki srdela iznosio je CF= 0, 699 ± 0, 043, dok je CF inćuna bio 0, 647 ± 0, 037. Rezultati dužinsko-masenog odnosa i kondicije srdele mogu se povezati s njenim završetkom mrijesta. Također, dobivene vrijednosti istih pokazatelja kod inćuna upućuju na pripremu populacije za nadolazeći mrijest.

Since conventional statistical techniques cannot analyze large numbers of interrelated variables, a statistical approach with the power to extract principal components from a multitude of data needs to be applied. Application of mathematical and statistical methods used in order to process large data sets in an optimal way is termed multivariate data analysis. Multivariate tools (MVA) used for reduction of variables, for classification and grouping, are Factor Analysis (FA) and Principal Component Analysis (PCA). They can give an overview of complex multivariate data and reveal relations between observed samples, allowing detection of significant variables as well as outliers. The aim of the study thus was to assess the value and applicability of MVA for hematological and plasma biochemical responses of fish over two seasons, using physicochemical water properties and heavy metals concentration of water as determinants of multiple fish stressors.

Bioactive potential of brown alga Padina pavonica from the Adriatic Sea was described according to the tests conducted.

In the Northern Adriatic Sea, tuberculate abalone (green ormer) (Haliotis tuberculata L.) are particularly threatened by bottom trawling and seafood disturbance, and their populations face a decline. A low average depth of 30 m, and a weak bathymetric gradient characterizes the Northern Adriatic. The most important oceanographic factors in the Adriatic are seasonal salinity, the concentration of nutrients, light intensity, currents and temperature. Seasonal changes of oceanographic factors, including temperature, algae and wave motion, greatly affect the growth and physiology of green ormers. For this reason, the Northern Adriatic green ormer tissues were studied over the main seasonal periods for their microanatomical structure, biochemical and antioxidant defense properties. Tissue concentrations of glucose (GLU), triglyceride (TRIG) and cholesterol (CHOL) were determined as the main energy sources for metabolic demands. The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), total antioxidant status (TAS) were evaluated as parameters for antioxidant capacity and lipid peroxidation. Tissue magnesium (Mg) and calcium (Ca) were investigated as formative shell elements, crucial for ionic homeostasis and reproduction control. Significant differences were found between spring/summer and winter samples for TAS, with peaks in June. GPx had the highest activities in fall samples, just as SOD, CHOL, GLU, TRIG, Mg and Ca parameters. Differences in green ormer tissue biochemical and antioxidant defense properties over seasons are shown in the figure as a log10-based scale. Data were grouped tightly in ormer tissues in spring/summer and winter, having unsymmetrical interquartile ranges. Long-term temperatures between 8.5 and 9.0°C are the lethal limit for ormers, and at low temperatures, they are limited in their ability to absorb food and grow. Winter low temperatures thus contributed to lower TAS and SOD concentrations. Not only antioxidant capacity, but also tissue Mg, Ca and GLU were on the decreased levels in ormers in winter. However, in fall a number of parameters had their yearly peaks, such as Mg, Ca, SOD, GPx, TRIG, CHOL and GLU. Interestingly, in this work ormers had elevated total antioxidant status in the warmer period, which can be attributed to the availability of food containing antioxidant compounds. During the spring/summer season, gonads were at the mid-maturation stage, and in fall, the ripe and spawned stages were recorded.

The present study was undertaken to evaluate the toxic potential and novel in-vivo antioxidant activity determination of polysaccharide fractions and to complete this research by developing a robust platform for the high-throughput screening of novel marine compounds with antioxidant properties.

The main objective of this work was to identify the most common bacterial strains in the Nin peloid mud, their biochemical profile, and their fatty acid composition.

In order to investigate the sensibility and accuracy of MALDI-TOF MS identification of S. baltica strains from Prussian carp, the isolates were grown on two culture media enriched by various NaCl concentrations, incubated at different temperatures and incubation times

Seasonal variation of biochemical and antioxidant parameters in scallop Pecten jacobaeus tissues were investigated.

Investigation of the electrochemical behaviour of astaxanthin using voltammetry of immobilized microdroplets and its application to determine the carotenoid concentrations in shrimp and soft-shell crab samples that are common in the human diet.

Antimicrobial and embryotoxic activity of brown algae Padina pavonica from the Adriatic sea were investigated.

The present study was undertaken to evaluate the environmental impact of olive mill wastewater (OMW) to living organisms, to identify the OMW components among phenolic group which are directly correlated with OMW’s toxicity and to complete this research with pointing out a huge potential of their use as a renewable resource.

The objective of this study was to evaluate the potential application of sodium selenite as neutral lipid inductor in marine microalgae Dunaliella tertiolecta which could favor the production of potential microalgae derived- biodiesel.

Mikroalge su prokariotski ili eukariotski fotosintetski mikroorganizmi jednostavne stanične građe koji predstavljaju bogat izvor karotenoida, vitamina i fenola. Dobre karakteristike mikroalgi, poput brzog rasta uz korištenje ugljičnog dioksida, visokih prinosa biomase, sposobnosti skladištenja esencijalnih hranjivih tvari i mogućnosti primjene genetskih modifikacija, osnova su njihove široke primjene u različitim granama industrije. Mikroalge mogu sintetizirati antioksidativne molekule (npr. polifenoli) koje sprječavaju peroksidaciju lipida i time produljuju rok trajanja prehrambenih proizvoda. Nedovoljno literaturnih podataka o antioksidativnoj aktivnosti polifenola iz ekstrakata mikroalgi predstavlja prepreku njihove uporabe u prehrambenoj industriji. Glavni cilj ovog istraživanja bio je utvrditi antioksidativnu aktivnost ekstrakata morske mikroalge Dunaliella tertiolecta ovisno o načinu sušenja, predtretmanu i vrsti ekstrakcije. Za svaki od ekstrakata određen je ukupan udio polifenola i njihova antioksidativna aktivnost. Prema rezultatima, udio polifenola i kapacitet antioksidativne aktivnosti zabilježen na uzorcima liofilizirane biomase mikroalge D. tertiolecta veći je u odnosu na uzorke komercijalno dostupnih slatkovodnih mikroalgi (Chlorella vulgaris). Dobiveni rezultati dali su uvid u optimalne uvjete ekstrakcije mikroalge D. tertiolecta i ukazali na njezin potencijal primjene u prehrambenoj industriji. Istraživanja su pomogla u razumijevanju postupaka ekstrakcije i iskoristivosti dobivenih ekstrakata mikroalgi u stvaranju ekonomski održivog i isplativog proizvodnog postupka sinteze prirodnih antioksidansa.

The sustainable management of fisheries and processing industry, as well as competitive development of aquaculture, are crucial for supplying quality fish products within the EU. Marine biotechnologies are estimated to grow 12% per year and the added value of bioeconomy approach to these sectors are fisheries management, aquaculture development, food waste, by-products, seafood safety and blue biotechnologies for food and non-food use. New innovative products could be brought to the markets by promoting further investigations on marine biotechnology. Marine biotechnology thus is interrelated with the bioeconomy sector and is recognized as the key engine for bioeconomy development. With that in mind, the European strategy for innovation for sustainable growth: A bioeconomy for Europe was enacted. Various strategies and programs still need to be developed at different levels and aligned with broader bioeconomy goals. Also, the understanding of marine biotech landscape needs to be improved, as well as ways to stimulate development from basic science to commercial application. However, development should be secure and sustainable, protecting the marine environment and its resources.

Treated wastewaters are abundant with complex pollutants having a significant impact on aquatic ecosystems. Since fish respond to low concentrations of environmental pollutants, they are ideal models for assessing these effects. The biological effects may affect all levels of biological organization, and it is thus a challenge to identify adequate biomarker indicators of toxic effects, especially since the treated wastewater effluents are chemically complex, constantly changing and biologically active (Topić Popović et al., 2016). Prussian carp (Carassius gibelio) is the most dominant fish species in lentic and slowly running aquatic habitats (Lusk et al. 2010). Several carp species have previously been included for in situ evaluation of effects of wastewater treatment plants (WWTP), but their impact on native species is less investigated, although assessment of biological responses of wild-caught fish in WWTP-related waters will more likely indicate actual ecological effects of treated wastewater. The scope of this work was thus to assess the impact of treated wastewaters on native wild Prussian carp inhabiting respective waters, with a hypothesis that fish would display pronounced biological responses. To that end, general fish health status was determined, as well as an array of selected plasma biochemical, haematological, oxidative stress and tissue histopathological indices.

Antioxidants are valuable molecules capable to inhibit possibly harmful oxidation, a chemical reaction whereby produced free radicals can damage cells and cause different diseases. To prevent cellular damage, marine and freshwater microalgae have developed antioxidant defence mechanisms and have attracted considerable attention as a promising source of natural antioxidants to replace synthetic antioxidants for food and chemical industry applications. The main goal of this study was to determine the difference in potential antioxidant capacities of marine algae Dunaliella tertiolecta and freshwater algae Chlorella vulgaris using fast electrochemical method named voltammetry of immobilized microparticles. This method allows determination of electroactive compounds in crystalline state without prior preparation of the sample. Aliquots of powdered dried algae were placed on a ceramic tile forming a spot of finely distributed material. Afterwards they were abrasively transferred to the surface of paraffin impregnated graphite electrode by rubbing the electrode rod with its lower circular surface on that spot of the sample. Measurements were preformed using cyclic and square-wave voltammetry in 0.1 M KNO3 on both tested samples with changing the experimental conditions (scan rate and frequency) for both techniques. In addition, changing the pH- value of electrolyte gave insight into to electrochemical mechanism of tested samples. For tested algae, different results were obtained which corresponds to their different antioxidant capacity

Selenium (Se), essential natural trace element for various organisms recently attracts attention due to its potential beneficial effects in various fields, i.e. biomedicine, electronics and some other industrial applications. However, depending on the dose, it can exhibit adverse effects. Current opinion is that possible Se toxicity could be reduced if Se is applied in the form of nanoparticles. Despite this, little is known about possible negative impact of SeNPs on different organisms. In general, when addressing nanotoxicity, aquatic organisms are of major interest, since majority of engineered nanoparticles are released through waters into environment. In aquatic ecosystem microalgae acts as a major vector of Se transport from water to filter-feeders and other consumers. With the aim to determine safety of their use in cosmetic, food and pharmaceutical industry the influence of SeNPs on growth of freshwater microalgae Chlorella vulgaris and marine microalgae Dunaliella tertiolecta was compared to the influence of sodium selenite (NaSe2O3), inorganic Se source. SeNPs were synthesized by solution phase synthesis and characterized using powder X-ray diffraction. The hydrodynamic diameter and zeta potential of SeNPs were determined by dynamic and electrophoretic light scattering. Obtained results may contribute to the understanding of SeNPs toxicity and their biotechnological potential.

Selenium (Se) is a micronutrient metabolically required by a variety of organisms, including microalgae and bacteria. Chlorophyta (green algae) is a large group of aquatic photosynthetic organisms and about half of the global microalgal production corresponds to green microalgae (Chlorella and Dunaliella) species. The range of Se concentrations within Se uptake goes from insufficient to toxic is very narrow. Se toxicity depends on its chemical structure and concentration and it is species- and dose- dependent in the case of microalgae. Recently, instead of sodium selenite (inorganic Se form), selenium nanoparticles (SeNPs) have obtained the attention as a possible Se source because of its lower toxicity. With the aim to determine safety of their use in cosmetic, food and pharmaceutical industry, the influence of SeNPs on growth and chlorophyll a content of freshwater microalgae Chlorella vulgaris and marine microalgae Dunaliella tertiolecta was compared with the influence of sodium selenite. Within this study, SeNPs were synthesized by solution phase synthesis and characterized using powder X-ray diffraction to examine SeNPs composition and phase. The hydrodynamic diameter and zeta potential of SeNPs were determined by dynamic and electrophoretic light scattering. These results provide a better understanding of the effect of SeNPs on green microalgae and their biotechnological potential, which may help in the development of new technological applications considering that nanotechnology becomes a promising tool in many fields of research such as pharmacy, environmental, information technology and engineering, chemical industries, and others.

The BIOECO-Research Driven Innovation project focuses on the core idea of the use of biomass from agricultural fisheries and forestry waste and residues with the purpose to foster green reconversion, multi-functioning, technology innovation, cross-sectoral integration. BIO- Economy aims at developing a Regional Innovation System for the Adriatic-Ionian area based on a structured bio- economy sector though the development of Research Driven Innovation (RDI) strategy at the regional and transnational level. It will improve the situation by: - creating a collaborative network among Adrion regions, enterprises and academia for collaborative research, knowledge transfer and skills development - supporting enterprises and clusters in the transition process toward an industrial model with higher level of innovation and international collaboration - boosting the integration between Green chemistry and Agri-food cluster according to the circular economy approach - creating an Adriatic-Ionian bio-based product market - bridging the gap among the existing wide regional disparities - activating a mutual learning process among regions with different levels of RDI and bioeconomy business maturity Project main outputs are: BIOECO-RDI strategies supporting regions in increasing bio-economy RDI level and cluster maturity, as well as BIOECO-RDI international network as the reference framework to develop innovation process.

Treated wastewater discharged from the wastewater treatment plant (WWTP) was evaluated by assessing its impact on downstream free-living fish (Prussian carp, Carassius gibelio) health status, microbiological contamination and antimicrobial resistance, fish tissue structure, blood biochemistry and erythrocyte nuclear abnormalities. Potential bacterial pathogens from fish and respective waters comprised aeromonads with a zoonotic potential. High resistance profiles were determined towards the tested antimicrobial compounds, mostly sulfamethoxazole and erythromycin. Histopathology primarily revealed gill lamellar fusion and reduction of interlamellar spaces of fish captured from the effluent. A significant increase in plasma values of urea, total proteins, albumins and triglycerides and a significant decrease in the activity of plasma superoxide dismutase were noted in carp from the effluent-receiving canal. A higher frequency of erythrocyte nuclear abnormalities was found in fish sampled from the effluent-receiving canal. Histological and haematological parameters differed significantly in effluent and downstream. Overall, treated wastewater modified the environmental parameters of the receiving surface waters, and played an important role in the antibiotic resistance scheme ; the investigated histological, hematological and plasma biochemical parameters of fish indicated to significant changes related to a complexity of environmental stressors. The histopathological changes observed in gill tissues in particular could be related to xenobiotics in the effluent, microbial load on gills, impaired water quality and increased heavy metal concentrations and may serve as an important end- point in the discrimination between polluted sites. There are potential public health concerns regarding aeromonad exposure amongst recreational fishermen who come into contact with fish inhabiting waters downstream from the WWTP, and WWTP workers who are occupationally exposed to wastewaters and their aerosols. A very high proportion of fish aeromonads demonstrated resistance, and a notable percentage of these exhibited a multiple resistance pattern. In this regard, negative effects of antimicrobial use on the environment, fish and human health should be elucidated, whilst more WWTPs should be involved in these complex studies.

Zeolite su industrijski najvažniji katalizatori u heterogenim procesima te je stoga ispitivanje njihovog utjecaja na okoliš nužno. Ovdje su predstavljeni rezultati istraživanja djelovanja nanočestica zeolita beta na embrije ribe zebrice.

Zeolites are widely used in various fields of human activities [1] and as such there are many particular cases when large amounts of zeolite powder can be released to certain area and have a potentially harmful effect on specific ecosystem. Therefore, the goal of this research is to prepare nanosized zeolite beta particles and evaluate their toxicity towards chosen representatives of vertebrates. For this reason zebrafish embryotoxicity test was performed [2] on two zeolite beta samples having different size. Zeolite beta samples have been prepared by hydrothermal treatment of alumosilicate precursors of molar oxide composition 0.35Na2O : 0.5Al2O3 : 25SiO2 : 9TEAOH : 295H2O at 100 °C 7 days and 1.23K2O : 1.23Na2O : 1Al2O3 : 50SiO2 : 25TEAOH : 745H2O at 140 °C for 30 h. The average size of synthesized zeolite Beta crystals was 140 nm and 640 nm, respectively. The washed samples have been freeze- dried and calcined at 550 °C. The solid product was characterized by XRD, DLS, TEM and TG. Prior testing, both merely dried and calcined powder have been dispersed in artificial water in three different concentrations. In addition, tetraethylammonium hydroxide solutions of corresponding concentrations have been prepared for the test. Standardized zebrafish Danio rerio embryotoxicity test has been conducted as follows: embryos have been exposed to zeolite beta suspensions up to 72 hours post fertilization (hpf), followed by determination of lethal (coagulation of the embryo, non- detachment of the tail, lack of heart beat and no somite formation) and sublethal effects (developmental retardation, deformations, formation of edems, blood accumulation, etc) of tested nanosized zeolites. In addition, at 48 and 72 hpf heart beat and hatching rate were evaluated, as well as pigmentation formation. All experiments have been performed on the non- protected embryonal stages, which do not require permission by animal welfare commissions. The collected data have been statistically analyzed. Results indicate the zeolites do exhibit a certain effect on the development of the Danio rerio embryos, but they are not lethal. The obtained results have showed that selected model organisms could improve our ability to understand the mechanism of the toxicity of alumosilicates, and should be incorporated in nanoparticles toxicity monitoring and risk assessment studies. Additionally, this research focuses on the importance of an early and accurate determination of the toxic potential of the nanoparticles to assess its further use.

Istražen je učinak pročišćenih otpadnih voda na zdravstveni status babuške (Carassius gibelio), mikrobiološku kontaminaciju i otpornost prema antimikrobnim lijekovima, strukturu tkiva riba, biokemiju plazme te nuklearne aberacije jezgre eritrocita. Među izoliranim bakterijskim patogenima značajan udjel činile su bakterije iz roda Aeromonas, potencijalni uzročnici zoonotskih bolesti. Najveća bakterijska otpornost pokazala se prema sulfametoksazolu i eritromicinu. U riba lovljenih u efluentu najčešći histopatološki nalaz bio je fuzija škržnih lamela uz smanjivanje interlamelarnih prostora. U plazmi istih riba uočeno je značajno povišenje vrijednosti uree, ukupnih bjelančevina, albumina i triglicerida, te značajno smanjenje aktivnosti superoksid dismutaze. Uočena je i učestalost pojave nuklearnih aberacija eritrocita, kao i značajna razlika histoloških i hematoloških parametara riba u efluentu u odnosu na nizvodne vode. Zaključno, pročišćene otpadne vode modificirale su okolišna svojstva recipijenata te su bile ključni čimbenik antimikrobne rezistencije ; histološki, hematološki i biokemijski parametri plazme riba ukazuju na značajne promjene koje se mogu korelirati sa okolišnim stresorima.

The need for sustainable aquaculture has prompted extensive research regarding the use of probiotics on aquatic organisms. Bacterium Lactobacillus rhamnosus was mixed in the trout diet with peptone water to attain two diet groups with concentrations of 10*7 CFU (G1) and 10*8 CFU (G2), respectively. The G3 feed group consisted of feed sprayed only with peptone water. First runway in line was stocked with the control group fed only the basal diet, second runway was stocked with G1, following with G2, and G3. They were fed for a period of six weeks and sampled at the beginning and end of the trial, and three weeks after the feeding trial. Before the trial, fish had no detectable lactic acid bacteria (LAB) in the intestines nor in the fecal contents. The numbers of viable lactobacilli recovered from fecal contents increased in G1 and G2 groups from below detection limits (

IMUNO-2865® is a natural hemicellulose compound with well-documented immunostimulative properties in people and marine mammals , but not yet tested on teleost fish. The aim of this study was to test in vivo effects of IMUNO- 2865® on blood biochemistry parameters in sea bream under aquaculture conditions during winter period. Blood Ca++ levels were significantly higher after 60 days of feeding in all treatment groups compared to the control, and remained elevated in group 4 even after 90 days following secession of supplementation. Antioxidant enzyme (SOD) was increased in group 4 compared to the control group from day 60 until the end of the experiment (P

Opisana je uloga i djelovanje probiotika, njihov utjecaj na ribe, načini uporabe probiotika. Opisani su rezultati istraživanja učinka Lactobacillus rhamnosus na kalifornijsku pastrvu.

Waste water treatment plants (WWTPs) are important point sources of a broad range of xenobiotics in the aquatic environment. Once released from WWTPs, xenobiotics represent a potential risk to environment, but also to human health. Since it is very hard to predict their possible impact on different levels of biological organization, it is necessary to use "early warning" biomarkers that can provide informations on potential environmental impact. Zebrafish embryos are frequently used organisms in ecotoxicology studies due to their short embryonic ontogenesis and transparent chorion. The fish embryo test (FET), as a substitute for acute toxicity with adult zebrafish, is a great biomarker in toxicity testing of pollutants that are residues from incomplete elimination in WWTPs. Screening sub-lethal and lethal endpoints after exposure to wastewater included coagulation of embryos, presence of somites, tail detachment, heart functioning, blood circulation, pigmentation, formation of edema and developmental retardation. Zebrafish embryos also possess multixenobiotic resistance mechanism (MXR) mediated by ABC transport proteins that acts as a protective barrier against the cell uptake and accumulation of potentially toxic compounds dissolved in the water. MXR mechanism can be modified by the presence of a wide range of xenobiotics frequently present in wastewaters (pharmaceuticals, chemicals used in industry and agriculture, personal care producs). Concentration of model fluorescent substrate in the embryo after exposure to environmental samples, give us an insight into the degree of inhibition and accumulation of xenobiotics from wastewater in the whole embryo. The integration of embryotoxicity and MXR creates a sensitive tool for the evaluation of early developmental responses of zebrafish to xenobiotics present in wastewaters on whole organism level, but also on the cellular level. These responses help us to understand better possible toxic effects of wastewater effluents on aquatic organisms.

Lactobacillus rhamnosus was mixed in the trout diet with peptone water to attain two diet groups with concentrations of 107 (G1) and 108 CFU (G2). The G3 group was fed with feed sprayed only with peptone water. Fish were fed for six weeks and sampled at the beginning and end of the trial, and three weeks after the feeding trial. Before the trial, fish had no detectable lactic acid bacteria in the intestines nor in the fecal contents. The numbers of viable lactobacilli from fecal contents increased in G1 and G2 from below detection limits (

Aquaculture is one of the worlds fastest growing food industries. There is, however, an over- dependence on the use of prophylactic and therapeutic antibiotics in aquaculture, with a significant cost to human and environmental health (bacterial resistance, residues, toxicity). Alternative products are being evaluated worldwide in the search for natural and cost-effective prophylactic supplements that can be incorporated into the fish diet. IMUNO-2865® is a natural hemicellulose compound with well-documented immunostimulative properties in people, terrestrial and marine mammals. The aim of this study was to test in vivo effects of IMUNO-2865® on blood parameters and oxidative stress responses in sea bream under aquaculture conditions. The research was conducted over a period of 6 months at a sea bream farm in Central Adriatic (Croatia). A total of 640 sea bream specimens (18 month old, weighing 277.8± 38.0 g) were divided into four groups, placed in four 8m3 net cages and conditioned for two weeks. Fish were fed with commercial pelleted food (Skretting) at a rate of 10 g dry diet/kg biomass/day. Four experimental diets containing 0 g (control Group 1), 1g (Group 2), 10g (Group 3) and 25g (Group 4) of IMUNO- 2865®/kg feed were prepared ahead of time and stored at 4oC. Samples were collected on days 0, 30, 60 and 90, after which the regular feeding regime was resumed at 15g dry diet kg-1 biomass. A final sampling was performed on day 180 (90 days after consumption of the last experimental diet). No mortality was observed throughout the experiment. Although blood parameters fluctuated among sampling periods (e.g. erythrocyte counts were highest at the end of the experiment), no significant differences were noted among treatment groups for total number of erythrocytes, leukocytes, basophils, eosinophils, lymphocytes and thrombocytes. However, a significant increase (P

Suspected vibriosis outbreak on sea bass farmed in floating cages needed confirmation and therapy/management instructions. A scheme comprising a combination of diagnostic and clinical methods was introduced for a better problem-solving. Within 24 hours since submission of fish for examination, bacterial cultures were isolated and rapid agglutination test identified V. anguillarum, while in 48 hours antimicrobial susceptibility was determined and targeted therapy recommended. Further physiological and biochemical tests were continued, which gave a readable result in 72 hours post submission of fish. Development of a rapid, sensitive and cost-effective protocol for identification of vibriosis caused by V. anguillarum is crucial due to quick onset of high mortality and great economic damage it can inflict.

The date mussel Lithophaga lithophaga (L.), is a well-known edible mussel. It is widely distributed in the Mediterranean Sea and on rocky areas of the eastern Atlantic, but knowledge of its biology and physiology is poor. The aim of this study is to show magnetic resonance imaging (MRI) technique as a practical hatchery tool, and provide the first MR imaging results for date mussel. The MRI technique is used to find a new way of date mussel exploring, with an emphasis on the preservation of life of the individual. This paper represents the beginning of using MRI in the study of protected species in Croatia. All MR images were performed on a SIMENS MAGNETOM Espree syngo MR B13 with 102 integrated matrix coil elements and up to 32 independent RF channels.

U nadzorima kvalitete konzervi sardina (Sardina pilchardus Walbaum, 1792) za potrebe Oružanih snaga Republike Hrvatske koji se obavljao na mjestu proizvodnje pretraženo je ukupno 27 uzoraka (n = 27) radi analize na prisutnost teških metala. Prosječna koncen- tracija olova iznosila je 0, 077±0, 0816mg/kg, kadmija 0, 0194± 0, 014 mg/kg. Količina žive (x) iznosila je 0, 067± 0, 0369 mg/kg, arsena 0, 775± 1, 006 mg/kg (n=27). Također u uzorcima konzervi (n=12), utvrđeno je prosječno 11, 054±9, 051 mg/kg cinka. Količina bakra je bila 1, 897±0, 826 mg/kg (n=11), dok je utvrđena prosječna koncentracija željeza iznosila 12, 73±6, 5 mg/kg. Od 27 uzoraka utvrđena je korelacija između količine olova i kadmija (r = 0, 75388, p

The amphipod Gammarus fossarum, stone crayfish Austropotamobius torrentium, an aquatic prosobranch gastropod mollusk Bithynia tentaculata, water from a pristine stream of the Nature Park Medvednica (Croatia), as well as scrapings from the sedimentary rocks, were sampled for microbiological evaluation. Rapid identification of environmental bacteria via commercial phenotypic tests allows for a wide choice of tests selection, and API 20E, an identification system for Enterobacteriaceae and other non-fastidious Gram-negative rods developed for clinical specimens, seems to be increasingly used for the identification of water-borne pathogens. However, due to several shortcomings of this system, such as the wrong identifications and the need of comparison with the diagnostic schemes based on reactions in conventional phenotypic tests, more advanced methods for identification are sought after. Therefore, a mass spectrometry MS-based method that focuses on the ribosomal proteins as a proteotypic system to utilize MALDI-TOF was used to achieve a highly accurate identification based on molecules that are translated from DNA but not subject to the expression variability seen in phenotypic methods. An excellent accordance of 57% was observed between the two methods in identification to the genus (Pseudomonas, Aeromonas, Shewanella spp.), while no correspondence was noted in identification to the species. Although this study furnished more accurate information on the bacterial populations in a surveyed freshwater stream (prevalence of Aeromonas and Pseudomonas spp.), and better clarified the existing relationships between the bacterial flora in tested samples, even a high-precision system had difficulties distinguishing between the species of the genus. Therefore, although implementation of MALDI-TOF MS can be a first step for identification which shortens the turnaround time, other methods for rapid analysis are warranted in order to obtain comparison of data and achieve an unambiguous identification.

In many organisms, the multixenobiotic resistance mechanism (MXR) is mediated by the activity of ABC transporters that bind and actively effl ux different toxic substrates from cells. This study attempted to identify MXR-related genes in the earthworm Eisenia fetida and compare the impact of the selected modulators, including verapamil, cyclosporine A, MK571, probenecid, orthovanadate, bisphenol A, and snow samples on the earthworm’s MXR. To confi rm the presence of MXR-reversing agents in E. fetida, we measured the accumulation of model substrates rhodamine B and rhodamine 123 in the body tissue of an adult earthworm. Experiments performed using the fi lter paper contact test showed that all of the tested modulators and environmental samples signifi cantly inhibited transport activity of the model substrates. Furthermore, partial mRNA sequence coding for P-glycoprotein (P-gp/Abcb1) was identifi ed and showed a high amino acid identity (up to 78 %) with homologues from different organisms. We could say that the ABC transporters are involved in various specifi c mechanisms that help earthworms to survive in a polluted environment and that their effi ciency can become compromised by environmental contamination.

ENA test se temelji na utvrđivanju učestalosti pojave eritrocita s jezgrama nepravilna oblika. Cilj rada bio je prikazati upotrebu ENA testa u genotoksičnim istraživanjima, te usporediti razine spontanih i induciranih morfoloških promjena eritrocitnih jezgri kod različitih vrsta riba. Rezultati su pokazali da postoje značajne razlike kako između tako i unutar samih vrsta. Razlozi varijabilnosti mogu biti vezani za specifičnost vrste, abiotičke čimbenike koji utječu na fiziološke procese, različiti kriteriji u klasifikaciji jezgrinih morfoloških promjena, te tumačenje rezultata testa. Stoga su potrebna daljnja validacijska istraživanja uz jedinstvene kriterije provođenja ENA testa kako bi se utvrdile referentne vrijednosti te se na taj način objektivizirala procjena genotoksičnih učinaka.

One of the most intriguing cellular defence strategies evolutionary developed in aquatic organisms is the activity of the multixenobiotic resistance (MXR) mechanism first described in early 1990s. Analogous to the well-known multidrug resistant (MDR) mechanism discovered in tumour cells, MXR in aquatic organisms is mediated by expression of the same transmembrane ATP-dependent proteins (ABC proteins). MDR/MXR results from the rapid efflux of a wide variety of potentially toxic xenobiotics out of the cell. The best-studied ABC protein in aquatic organisms is the P-glycoprotein (ABCB1), while recent studies demonstrate the importance of other types of transport proteins such as MRP (ABCC) and the presence of the MRP-related genes in fish and mussels. The main goal of this study was the detection of ABCC (MRP) related gene sequences and functional detection of Pgp and MRP-like transport activities in aquatic animals. These new findings should help in better characterization and identification of all proteins possibly involved in MXR phenomenon and will influence afirmation of these biomarker in evaluation of environmental quality, especially in aquaculture.

Numerous pharmaceuticals are metabolized by P450 enzymes in fish, which makes them of critical importance both for detoxification and formation of toxic metabolites. Therefore, a better understanding of the role of each cytochrome P450 in drug metabolism and drug-induced toxicity is vital. At this time, however, most research on the P450 system in fish has been done on environmental factors that influence P450 induction. Much less is known about metabolism of aquaculture antibiotics by the cytochrome P450 system. Despite strict regulations, except via medicated feed, fish may get exposed to antimicrobial drugs via aquatic environment. Discharge from sewage treatment plants has been identified as their primary source. The differences in responses of CYP enzymes in different fish species to antimicrobial treatment may have relevance for the use of antimicrobials in aquaculture. The variable effects in the response among different fish species to antimicrobial drugs is a valid reason to critically use assays developed for mammals for evaluating drug response in various fishes. Also, the use of antibodies to CYP enzymes across phyla can be of value if caution is exercised in the interpretation. Substrates developed for use in mammalian systems have been assayed in fish, and are useful for CYP-mediated functional comparison between mammalian and fish species. Standardized protocols for drug impact on fish P450 will facilitate those studies and standards will be necessary for the future application on fishes.

Tricaine methane-sulfonate (MS-222) is one of the most widely used anaesthetics for poikilotherms worldwide. This paper outlines its anaesthetic efficacy and dosage in fish and legislation for its use, fish stress responses to MS-222 anaesthesia, its effect on fish physiology and blood properties, pharmacokinetics, genotoxicity, immune response, potential interference with fish hepatic cytochrome P450 spectra, and its impact on nerve sensitivity. The key questions arising from the available data are analyzed, such as regulatory constraints on its use, the need for the standardization of buffering protocols, and interdependencies of the factors impacting the specific applicative efficacy of MS-222. Current research has provided an abundance of data on MS-222 use in fish, though the applications within these studies are often impractical at the farming level. Therefore, specific emphasis is placed on highlighting application strategies on a practical basis and presenting potential future research on topics that require in-depth analysis (preparation and storage of anaesthetic solutions, preanaesthetic sedation and stress reduction, cortisol response in aquarium fish, toxicity of MS-222 metabolites, and possible immunodepressive properties). Additionally, both from a scientific and practical perspective, we must have a better understanding of its safety margins, induction, immersion and recovery times for many (marine and freshwater, farmed and ornamental) fish species in order to achieve optimal utilization.

Antibiotic nitrofurantoin (NF) and its derivatives are used in veterinary and human medicine. The impact of NF on the aquatic environment is largely unknown and its ecotoxicity is still under investigation due to conflicting opinions about its use. Increasing evidence suggests that NF can cause various adverse effects in living cells. In this investigation, we used three different fish cell lines (PAC2, PLHC-1, R1) to evaluate cytotoxicity of NF toward fish. PAC2 cells were the most sensitive to NF by MTT colorimetric assay, exhibiting an EC50 of 15 µM (3.57 mg/L) NF. Growth inhibition of unicellular algae Scenedesmus subspicatus and mutagenic action towards Salmonella strains (TA 98 and TA 100) using classical Ames test were also determined. Significant decrease in growth of unicellular algae after 24 h exposure to NF (1, 10, 30, 100, 200 µM) was observed. Mutagenicity test responses revealed a large number of revertants following exposure of NF. These results indicate a substantial risk of NF to the health of aquatic ecosystems.

The composition of tail muscle fatty acids from wild and cultured bluefin tuna reared on a diet based on herring and sardine, along with the plasma lipid profile of the farmed individuals, was determined. The total lipid content of farmed bluefin in this study was 0.922 g/100 g, or 3.49 g of SAFA, 4.48 g of MUFA, 2.58 g PUFA n-3, and 0.37 g of PUFA n-6 fatty acids ; for wild specimens it was 0.920 g/100 g, or 2.85 g of SAFA, 4.82 g of MUFA, 2.78 g PUFA n-3, and 0.27 g of PUFA n-6 fatty acids. Major fatty acids in this study were 16:0 ; 16:1, n-7 ; 18:1, n-9 and DHA 22:6, n-3 acids. The sum of these major components accounted for more than 57 % and 80 % of the total fatty acids in all samples of farmed and wild tuna, respectively. No significant differences in proximate composition were demonstrated between farmed and wild samples, except for energy value, in favor of the farmed tuna. Statistically, GLU tends to increase together with CHOL and TRIG, since for these pairs it showed positive correlation coefficients and P > 0.05. Some measured tuna metabolites demonstrated strong mutual correlations, especially GLU, CHOL and TRIG, which are crucial factors in lipid profile of animals.

Fresh and frozen seafood were collected from a market in Croatia in warm/cold seasons and analyzed for their microbiological quality. Although constant control of seafood is provided by veterinary inspection, the main purpose of this work was to investigate the presence and the level of aerobic mesophilic bacteria, aerobic psychrophilic bacteria, Salmonella spp., Enterobacteriaceae, Escherichia coli, Staphylococcus aureus, sulphite-reducing clostridia, Listeria monocytogenes, Vibrio cholerae and V. parahaemolyticus in seafood presented for direct human consumption and to evaluate their possible variations regarding seasons. Most published work regarding microbial contamination of seafood concentrates on limited spectra of bacteria, mostly vibrios and coliforms, while this study provided a broader insight into microbiological indicators. Quality of samples varied between species and within seasons regarding total counts of aerobic mesophilic and psychrophilic bacteria. Surprisingly, 66.6 % of fresh and frozen fish were unacceptable. The overall prevalence of V. parahaemolyticus was 5 %. Its recovery rate was higher in fresh/frozen shellfish in both seasons than in other specimens or other storage/season conditions. Fresh crustaceans sampled in winter demonstrated significantly higher aerobic mesophilic counts than frozen ones. Unacceptable Enterobacteriaceae levels were obtained in 40 % of the summer fresh fish samples. The results of this survey constitute an indicator of bacteriological contamination of a variety of seafood and could serve as a basis for future testing of seafood, and possibly as a template for developing a regional/Mediterranean testing scheme on the microbial contamination of seafood.

Changes in erythrocyte nuclear morphology have been increasingly used as complementary test to micronucleus (MN) assay as a common protocol to evaluate genotoxic effects especially in aquatic organisms such as fish. Occurrence of erythrocyte nuclear abnormalities (ENA) and MN are detected also in other non-mammalian species with nucleated erythrocytes such as birds, amphibian and some reptilian species. However, prior to including them in the regular assessment, it will be necessary to determine baseline values and confounding factors which may influence the level of spontaneous MN and ENA appearance, and to determine to what extent their presence can be inherent to certain species. The aim of this study was to compare their level and seasonal variability between fish (represented by sea bass Dicentrarchus labrax, and sea bream, Sparus aurata) and reptile-snake (represented by Balkan whip snake, Hierophis gemonensis). Light microscopic examination of the specimens revealed appearance of the same type of erythrocyte nuclear changes in both examined species groups. Summarized data showed significantly higher frequency of ENA and significantly lower MN in snake than in fish. Interspecies comparisons revealed that the lowest NA values were recorded in the spring in both groups. However, the highest level was reached during the winter in snakes, and autumn in fish. In fish, frequency of micronucleated erythrocyte has the same seasonal pattern as ENA. The most frequent MN appearance in snakes was detected in winter, and the lowest one in spring. Despite that the MN frequency does not exactly match ENA pattern during the seasons, statistical analysis revealed significant positive correlation between ENA and MN.

Plasma biochemical profile was examined in cage cultured shade fish Argirosomus regius in order to evaluate physiological status and provide detailed picture of its seasonal changes. Biochemical procedures were employed to study trygliceride (TRIG), cholesterol (CHOL), total protein (TP), glucose (GLU), urea (BUN), inorganic phosphate (PHOS), calcium (Ca), creatine kinase (CK), lactate dehydrogenase (LDH), ammonia (NH3), magnesium (Mg), aspartate aminotransferase (AST), amylase (AMYL), alkaline phosphatase (ALKP), creatinine (CREA), gamma glutamyltransferase (GGT) and total bilirubin (TBIL). The results were evaluated through different statistical analyses. Conventional statistical methods do not provide solutions that meet the goals of the research, so machine learning methodology was used to construct and evaluate classifiers as well as to assess parameter (plasma metabolites and enzymes) importances with respect to the target variable (season). Two machine learning methods, decision tree algorithm and PARF - a new implementation of Random Forest algorithm were applied to the problem of seasonal fish population modelling based on blood samples data collected. These two methods were used in order to explain which combination of parameters best demonstrates the differences between examined seasonal groups through construction and evaluation of classifiers and using PARF approach to the assess the variable importances with respect to the target variable, i.e. season. It is evident that about half of the parameters (UREA, ALKP, NH3, CHOL, Ca, PHOS, GLU and CREA) have significant importance (i.e. exhibit significant differences between seasons) for seasonal discrimination models. From our classifier learning experiments it is evident that using only 2 (UREA, ALKP), out of 8 high importance ranking parameters (UREA, ALKP, NH3, CHOL, Ca, PHOS, GLU, CREA) is generaly enough to obtain high accuracy classsifiers, which suggests rather high correlation between parameters on a seasonal scale. We found that AST, Ca, TP, LDH, BUN, CREA, AMYL, CK, NH3 were increasing with elevation of water temperature (measured in March, May and July) while CHOL and ALP were decreasing at the same time. AST, TP, GLU are sensitive indicators ; a rise of their activity can be recognized during metabolic modification related to nutritive imbalance (occuring in the colder season) resulting in physiological dysfunction or cellular integrity. ALP is a splitting enzyme of terminal phosphatase groups from organic esters found mainly in the hepatocites. High values are indicative of liver, specifically biliary impairment. CREA and BUN levels can indicate other problems besides those intrinsic to the kidney such as prerenal problem. CHOL could be nutritionally related and it is possible that food intake are higher in the summer compared to the winter. Ca levels were lower in the period of cold water this might be due to perturbations in the hormones during that control Ca. Decrease ALP levels associated with conditions linked to the biliary tract. According to One Way Analysis of Variance (Sigma Statistical Software ver. 2.01), TRIG and LDH were not significantly different between seasons, while CHOL, BUN, Ca, NH3, AST, ALKP and CREA demonstrated significance in all sampling points. Three parameters (TP, PHOS, and AMYL) were significant between March-July and May-July ; while GLU, CK and GLU, Mg showed significance for period March-May and March-July, respectively.

Live lobsters Palinurus elephas (Fabricius, 1787) were collected from a number of sites around the island of Vis, and transported to laboratory exposed to air for considerable time. The aim of the study was to examine the effect of emersion on the plasma biochemical profile which is a useful indicator of physiological status. Lobsters had average body weight of 517, 09 g and carapace length of 113, 20 mm. Haemolymph was withdrawn from ventral abdominal artery. Biochemical procedures were carried out to study metabolites (trygliceride, TRIG ; cholesterol, CHOL ; total proteins, TP ; glucose, GLU ; urea, BUN ; creatinin, CREA) and several enzymes (alkaline phosphatase, ALKP ; aspartate aminotransferase, AST ; gamma glutamyltransferase, GGT ; lactate dehydrogenase, LDH). In addition to classic statistical analyses, six machine-learning methods utilizing ten times ten-fold cross-validation evaluation were used in order to determine the best classification model for the blood biochemistry data. Blood GLU, BUN, ALKP, TP significantly increased while CREA was significantly lower. This approach clarifies the role and importance of physiological processes which show diversity of functional characteristics of various biochemical parameters in case of long term exposure to air.