Search

Coastal zones of the Adriatic Sea are continuously subjected to multiple anthropogenic pressures including pollution from urban areas, industry, agriculture and aquaculture, habitat degradation, introduction of invasive species and climate change. To preserve its integrity, countries within this region, including Croatia, have adopted a number of initiatives. One of them is the Marine Strategy Framework Directive (MSFD) developed with the aim to monitor and preserve Good Environmental Status (GES) of the Adriatic Sea. Even though multiple anthropogenic pressures are affecting all components of these ecosystems, MSFD overlooks the importance of human-induced changes on the level of complex microbial communities, having key role in functioning of marine food webs, biogeochemical cycling and in maintaining stability and health of marine ecosystems. To tackle this issue project MicroLink, funded by the Croatian Science Foundation, is intended to find that missing link that exists between changes in microbial assemblages and ecosystem quality, paving the way towards integration of microbes among qualitative Descriptors of the GES. Altogether, 67 sediments were collected from seven harbors situated along the eastern Adriatic coast, being defined as pollution hot spots in the Adriatic, and a comprehensive physicochemical characterization was conducted (measuring concentrations of selected metals, Hg, TP, TN, TC, TBT, toxicity…). To determine responsiveness of benthic microbial assemblages to the observed pollution we investigated changes in microbial communities on several levels including (i) structural changes (in bacterial, archaeal, protists and fungal communities), (ii) changes in interaction networks and (iii) functional changes (abundance of important biogeochemical cycling genes). We believe that changes observed on the level of benthic habitat, being a sink for accumulation of pollutants, can provide us with valuable insight into the long-term consequences of anthropogenic pressures on the Adriatic environment, with microbes that could potentially be used as an early warning sign of anthropogenically polluted environment.

Considering the major anthropogenic pressures affecting marine ecosystems, assessment of the “health status” of the coastal zones is an important topic, especially considering that 50% of the world population inhabits those areas. There has been a number of directives implemented by Mediterranean countries regarding protection and preservation of marine coastal areas. Unfortunately, none of them mention the potential contribution of microorganisms in marine ecosystems health quality assessment. Microorganisms play pivotal role in functioning of the whole marine ecosystem by mediating biogeochemical cycling of all major elements, C sequestration and elimination of pollutant. In the same time microorganisms are characterized by their fast responsiveness to environmental changes, hence anthropogenically-driven pressures in coastal areas could cause changes on the structural and functional level of microbial communities, eventually altering the trophic structure of the whole food web. Through the multidisciplinary and innovative approach, using different molecular tools, MicroLink project, financed by the Croatian Science Foundation, aims to emphasize the need to define potential contribution of microorganisms as quality indicators of marine ecosystems health. Sampling of the sediment material was performed in seven different harbors settled along the Adriatic coast, which represent sites under multiple pollution pressures. Comprehensive physico- chemical characterization of the sediment was used for their pre-classification in different groups thus representing sediments with different “anthropogenic disturbance gradient”. This data sets were further correlated with benthic microbial assemblages data obtained by the multi- layer, multi-trophic molecular-based analyses (structure/diversity/function of the benthic bacterial/archaeal/fungal/protists communities). MicroLink project is going to provide guidelines for future monitoring of anthropogenic pressures in coastal zones in order to improve marine environment policies.

The surface of brown macroalgae is colonized by epiphytic bacteria which are characterized by abilities of fast colonization, quick metabolic response to algal exudates, and adaptation to environmental variations. Epiphytic bacteria found on Fucales differ significantly from bacterial communities found on surrounding abiotic surfaces [1, 2]. In addition to the fucacean ecology and the vegetative cycling that significantly influence species resilience to the climate and anthropogenic stress, positive species interactions in the phycosphere can act as a first defense barrier against abiotic stress [3]. The study aimed to determine epiphytic bacterial communities of selected fucacean species (Treptacanta barbata, Carpdesmia crinita, and Cystoseira compressa) and to explore the relationship between the fatty acid profiles of selected fucacean species and their associated epiphytic bacterial communities. The thalli of sampled species were divided into perennial and annual parts. T. barbata and C. crinita show high contents of mono- and polyunsaturated fatty acids. The branches and apical parts of C. crinita demonstrated high proportion of both C18 and C20 polyunsaturated fatty acids but also, C. crinita showed the highest unsaturation degree (4.32%) in opposition to the highest proportion of saturated fatty acids which was found in thalli of C. compressa. Several representatives of the Gammaproteobacteria class, identified as Vibrionales, Cellvibrionales, and Xanthomonadales were found which contributed strongly to the separation of T. barbata apical parts and branches, C. compressa receptacles and C. crinita branches from the T. barbata cauloids and C. crinita apical parts and cauloids. The results showed a strong relationship between fatty acid profiles of the analyzed species and phycosphere community structure, underlining the host physiological condition in shaping the biological interactions and maintaining a healthy microbiome.

Harmonization of monitoring protocols and analytical methods is a crucial issue for transnational marine environmental status assessment, yet not the only one. Coherent data management and quality control become very relevant when environmental status is assessed at regional or subregional scale (e.g., for the Mediterranean or the Adriatic Sea), thus requiring data from different sources. Heavy metals are among the main targets of monitoring activities. Significant efforts have been dedicated to share best practices for monitoring and assessment of ecosystem status and to strengthen the network of national, regional and European large data infrastructures in order to facilitate the access to data among countries. Data comparability and interoperability depend not only on sampling and analytical protocols but also on how data and metadata are managed, quality controlled and made accessible. Interoperability is guaranteed by using common metadata and data formats, and standard vocabularies to assure homogeneous syntax and semantics. Data management of contaminants is complex and challenging due to the high number of information required on sampling and analytical procedures, high heterogeneity in matrix characteristics, but also to the large and increasing number of pollutants. Procedures for quality control on heterogeneous datasets provided by multiple sources are not yet uniform and consolidated. Additional knowledge and reliable long time-series of data are needed to evaluate typical ranges of contaminant concentration. The analysis of a coherent and harmonized regional dataset can provide the basis for a multi-step quality control procedure, which can be further improved as knowledge increases during data validation process.

River estuaries and semi-enclosed bays with poor water exchange are considered as sensitive coastal areas. The determination of nutrients and potential toxicity in sediments represent important elements in their environmental quality assessment process. Sediments act both as sink and as a source of toxic environmental compounds. In this study, spatial variations of sediment nutrients and toxicity in coastal areas with limited water exchange (Rječina River basin, Bakar Bay) in vicinity of the industrial and urban centre of Rijeka was examined. In sediment of Rječina basin, both total nitrogen and total phosphorus exhibited a clear spatial pattern characterized by the natural and anthropogenic enrichment of the sediment along the river flow. The N:P ratio ranged from high upstream to low in marine sediments. Potential sediment toxicity varied from highly toxic to not toxic, in accordance to the river flow and the anthropogenic input. In Bakar Bay, nitrogen and phosphorus originated from different sources. The main sources of nutrients were situated in the northern part of the bay while a higher homogeneity of environmental conditions was present in its southern part. No correlation between the concentration of nutrients and sediment toxicity was found in both investigated areas. Therefore, the local impact of different contamination sources created a heterogeneous scenario from both the geochemical and ecotoxicological point of view. Moreover, this study confirmed the Microtox® assay as a sensitive sediment toxicity screening tool and supported the idea of sediment toxicity as an independent parameter considered as vital in assessing ecological risks for biota and describing the environmental quality of sites with multiple sources of contamination.

Temporal variation in the level of acid DNase activity in gills and digestive glands of the mussels Mytilus galloprovincialis from pristine and polluted area were determined. Acid DNase activity is tissue specific and responsive to physiological requirements and environmental conditions. Preliminary data indicated acid DNase activity in mussel gills as promising biomarker of pollution.

New biomarkers of high sensitivity and low cost are widely investigated for marine biomonitoring purposes. Our previous investigations showed that the exposure of mussels to individual model marine pollutants causes the increase of acid DNase activity in hematocytes and hepatocytes indicating acid DNase activity as a promising biomarker. For further validation mussels were exposed to composite organic extracts of seawater from Adriatic (VRANJIC, industrial area – high potential toxicity, RIJEKA, harbor – moderate potential toxicity, PLOČE, small rural – low potential toxicity) and acid DNase activity was measured in gills and digestive gland. The patterns of influences in both tissues are site specific and related to different potential toxicity of sea-water extracts. In referent mussels acid DNase activity was higher in gills than in digestive gland. The extract from station with low potentional toxicity (PLOČE) did not induce statistically significant change the acid DNase activity in either tissue. Enzyme activity changed in gills of mussels exposed to extract with moderate potential toxicity (RIJEKA) and in digestive gland of mussels exposed to extracts with high potential toxicity (VRANJIC). In both cases enzyme activity increased already after 3 h, remained for 48 h and discriminate stations according to potential toxicity of organic extracts. Advantages/disadvantages of using acid DNase activity in mussel gills and/or digestive gland as biomarker of toxic pollutant presence are discussed.

New biomarkers of high sensitivity and low cost are widely investigated for marine biomonitoring purposes. Our previous investigations showed that the exposure of mussels to individual model marine pollutants causes the increase of acid DNase activity in hematocytes and hepatocytes indicating acid DNase activity as a promising biomarker. For further validation mussels were exposed to composite organic extracts of seawater from Adriatic (VRANJIC, industrial area – high potential toxicity, RIJEKA, harbor – moderate potential toxicity, PLOČE, small rural – low potential toxicity) and acid DNase activity was measured in gills and digestive gland. The patterns of influences in both tissues are site specific and related to different potential toxicity of sea-water extracts. In referent mussels acid DNase activity was higher in gills than in digestive gland. The extract from station with low potentional toxicity (PLOČE) did not induce statistically significant change the acid DNase activity in either tissue. Enzyme activity changed in gills of mussels exposed to extract with moderate potential toxicity (RIJEKA) and in digestive gland of mussels exposed to extracts with high potential toxicity (VRANJIC). In both cases enzyme activity increased already after 3 h, remained for 48 h and discriminate stations according to potential toxicity of organic extracts. Advantages/disadvantages of using acid DNase activity in mussel gills and/or digestive gland as biomarker of toxic pollutant presence are discussed.

In this study the microscopic and flow cytometric mesurements of micronuclei distribution in the tissue of the common marine bioindicator, mussel, Mytilus galloprovincialis after treatment with model pollutants were compared and discussed. Advantage of the flow cytometry detection of micronuclei distribution in mussel haemolymph after treatment with model polutants were presented.

The objective of this study was the assessment of seawater toxicity at 24 samoling sites along the Adriatic coast, Croatia, by use of a bacterial bioluminescence assay. Analyses of temporal changes during a seven year period (1999-2005) showed a decrease of potential toxicity (EC50) at 3 sampling sites and an increase at 2 sampling sites. The spatial distribution revealed that the region of Split, Kaštela Bay i sthe area with the heaviest load of toxic agents. The database created from the results of this study could serve in future as guidance of properly directed remediation activity and for the control of its efficiency.

The total concentrations of PAHs of 6 sampling sites of the Gulf of Rijeka vary from below detection limit to 303 ng/l in seawater, from 213 to 695 ug/kg DW in sediment and 49 to 134 ng/g WW in mussel tissue. Toxicity of seawater and sediment organic extract is correlated with PAHs content indicating that PAHs are predominant toxic compounds. There is a positive correlation between potential toxicity of mussel biological fluids and reduction of anoxic survival.

Our investigation based on the determination of potential toxicity of seawater organic extracts mirror the present status of seawater quality and background levels of potential toxicity at 24 selected sites along adriatic coast.

The quality of sea water along the Adriatic coast of Croatia was evaluated three times during 1.5 years at 16 sites located in protected, urban, industrial, harbor or brackish areas. Samples of sea water (30 l) were tested by the Microtox test for toxicity and the umu-test for genotoxicity. Samples of mussel gills (5 individuals, Mytilus galloprovincialis) were collected from each sampling site and the Fast Micromethod® was used to test DNA integrity, which may have been altered by water contaminants. According to the Microtox text, 75% of the water samples were potentially toxic. According to the umu-test, 40% were potentially genotoxic. Alteration of DNA integrity was detected in 40% of the mussel gill samples. Uzorci morske vode i škrge školjkaša uzimani su sa 16 lokacija zaštićenog i bočatog područja, kao i s područja s urbanim, industrijskim i lučkim utjecajem uzduž jadranske obale. Za određivanje toksičnosti koristio se Microtox test, za određivanje genotoksičnosti SOS/umu - test, te za određivanje cjelovitosti DNA molekule tzv. Fast Micromethod® test. 75% analiziranih uzoraka vode pokazalo je toksičnost, 40% uzoraka vode pokazalo je genotoksičnost, dok je 40 % uzoraka škrga dagnji pokazalo oštećenje DNA.

The potential toxicity of organic extracts from 12 seawater samples from each of 24 sampling sites, collected during 1999-2001 along the Adriatic coast, Croatia, was analyzed with the Microtox toxicity bioassay. The results were consistent with the usefulness of Microtox for the detection of accidental toxic events. To determine the water quality of selected areas, cluster analysis for discrimination between groups with similar toxicity load and water quality index as a base for the ranking of sampling sites was introduced. Based on our experimental data, five classes of the quality index were defined, and so areas were ranked in five categories (excellent, good, fair, poor, and very poor) according to their potential toxic influence. The water quality of selected sites for the potential toxicity of organic extracts could be described as excellent at one sampling site, good at 15 sampling sites, and fair at eight sampling sites. Poor and very poor seawater quality was not detected.

The potential toxicity of organic extracts from 12 seawater samples per 24 sampling sites, collected during 1999-2001 period along Adriatic coast, Croatia, was analyzed by Microtox bioassay. Discrimination between sampling sites according to their potential seawater toxicity was achieved by cluster analysis. The water quality of selected sites could be described as excellent at one sampčing site, good at 15 sampling sites and fair at 8 sampling sites

The quality of sea water along the Adriatic coast of Croatia was evaluated three times during 1.5 year (1998-1999) at 16 sites located in protected, urban, industrial, harbor or brackish areasd. 75% of the water samples were poptentialy toxic, 40 % genotoxic and alteration of DNA integrity was detected in 40 % of the mussel gill samples.

ard opisuje primjenu bioindikatorskih vrsta u moru za određivanje i praćenje učinka genotoksičnih zagađivala

The quality of selected species, fish, mussel, crab and sea cucumber as bioindicators for genotoxic impact is discussed

The expression and proteočytical cleavage of KRS_SD in control and TBT-treated sponge, Suberitas domuncula was studied. KRS_SD induction and cleavage during TBT stress suggest that in sponge cells exist mechanism similar to one present in human cells where proteolitically cleaved KRS/MST protein kinase is involved in promotion of apoptosis following environmental stress.

In order to clerify the suitability of mussel as bioindicator organisms for water quality determination general stres biomarkers (exposure to air and induction of heat shock proteins) and specific biomarkers (DNA damage and cell cycle alterations) have been tested. The results strongly support the suitability of mussels as a good bioindicator when selectedf biomarkers are used in biomonitoring studies

The effect of artificial and solar UV irradiation on DNA of marine phytoplankton was investigated.Artificial UV irradiation increases CPDs in dose-dependent manner. After sunlight exposure the level of DNA damage increase and efficient DNA repair mechanism was detected.

Zbog sjedilačkog načina života i ishrane filtriranjem morske vode, spužve se trenutno i neizbježno suočavaju sa svakom promjenom u svojoj okolini. Postojanje proteina KRS_SD, koji se u uvjtima stresa tributilnim kositrom i vodikovim peroksidom fosforilira i proteolitički cijepa od strane kaspazi-3 srodnog proteina ukazuje da u stanicama spužve postoji mehanizam odgovora na oksidativni stres sličan onom u humanim stanicama, u kojem protein kinaza MST/KRS sudjeluje u promociji apoptoze

UV-absorbing compounds production is responsive to illumination regime that is consistent with the protection role of UV-absorbing compounds. Exposure to artificial UVB showed a clear 24 hr repair pattern in I. galbana DNA. No significant influence on CPDs removal was detected in samples recovering under constant illumination in the presence of DNA repair inhibitors (caffeine or nalidixic acid). When cells grown under 16h light : 8hr dark regime were recovering in the dark (no photorepair) and in the presence of nalidixic acid (no nucleotide excision repair) the initial amount of CPDs in DNA remained after 24 hours. I. galbana cells posses DNA repair mechanisms that are efficient in CPDs removal during 3 hrs of sunlight exposure.

Phytoplankton cultures grown under 16/8 light/dark regime contained smaller amount of photosyntetic pigments and UV absorbing compounds and showed greater sensitivity to UV light then the cultures grown under 24/0 light regime

A rapid and convenient procedure for DNA damage determination in cell suspensions and solid tissues on single microplates was developed. The procedure is based on the ability of commercially available fluorochromes to interact preferentially with dsDNA in the presence of ssDNA, RNA, and proteins at high pH (>12.0), thus allowing direct measurements of DNA denaturation without sample handling or stepwise DNA separations. The method includes a simple and rapid 40-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 12.4 after NaOH addition. The time course and the extent of DNA denaturation is followed in a microplate fluorescence reader at room temperature for less than 1 h. The method requires only 30 ng DNA per single well and could conveniently be used whenever fast analysis of DNA integrity in small samples has to be done, e.g., in patients' lymphocytes after irradiation or chemotherapy (about 3000 cells per sample), in solid tissues or biopsies after homogenization (about 25 mu g tissue per well), or in environmental samples for genotoxicity assessment.

Ser/Thr like kinase from the marine sponge Suberites domuncula, termed STE20Sub has been cloned and its aminoacid structure characterized. The catalytic domain is most homologous to STE20 and other STE20 like kinases, and the whole molecule shows the highest homology (similarity) to Krs-1,2 and Mst-1,2 which are human stress responsive kinases. Different levels of kinase expression are expected in sponges under the influence of different environmental contaminats in relation to nonstressed sponges.

The presence of cyclobutane pyrimidine dimers in unlabelled phytoplankton DNA was detected by a simple alkaline filter elution assay and T4-endonuclease V digestion. The quantity of dimers per genome was estimated by alkaline agarose gel electrophoresis. The techniques were used to detect and quantifiy UV induced damage and repair in phytoplankton Isochrysis galbana DNA. Repair of cyclobutane pyrimidine dimers induced by artificial UV and natural sunlight was altered in the presence of water soluble crude oil fractions. These results indicate that phytoplankton communities could be more susceptible to UV damage in areas with antropogenic pollution.

Učinak UV-zračenja na DNA morske fitoplanktonske vrste Isochrysis galbana promatran je u laboratorijskim uvjetima i nakon izlaganja sunčevom svjetlu. Mjerena je učestalost ciklobutanskih pirimidinskih dimera (CPD-a) metodama alkalnog filtar eluiranja i alkalne gel elektroforeze koje su prilagođene radu s enzimom T4-endonukleaza V i stanicama fitoplanktona. Stanice fitoplanktona koje su imale 24/0 satni ritam svjetla i mraka u odnosu na stanice s ritmom 16/8 sati su manje osjetljive na UV-zračenje zbog povećane koncentracije fotosintetskih i UV-apsorbiraju ih pigmenata. Zabilježen je linearni porast CPD-a s porastom doze umjetnog UV-zračenja za obje kulture.Tijekom 24 sata nakon zračenja, uz popravak CPD-a od 70-95% usporava se i rast kulture. Nakon izlaganja kulture prirodnom sunčevom zračenju povećava se broj CPD-a ali i njihov učinkoviti popravak, mjerljiv tri sata po izlaganju. Učinkovitost popravka DNA se smanjuje uz prisutnost 10% vodotopive frakcije nafte u morskoj vodi uzrokujući stalni porast broja mjerljivih oštećenja. Uzajamni utjecaj UV-zračenja i vodotopive frakcije nafte rezultira smanjenom sposobnošću rada mehanizama popravka DNA fitoplanktona I. galbana

Increased level of UV was evidenced recently. Because of its natural exposure to sunlight phytoplankton has become an important object for determination of UV effects on living species. The aim of our study was to investigate the effect of UV irradiation on cell genome and DNA in marine phytoplankton I. galbana . Phytoplankton monocultures were irradiated under laboratory conditions or were exposed to sunlight. The effect of UV was measured by determination of cyclobutane pyrimidine dimers(CPDs) that represent an exclusive and specific DNA damage induced by UV. CPDs are formed in DNA by covalent interaction of two adjacent pyrimidines in the same polynucleotide chain resulting in lesion that can interfere transcription and DNA replication and could finally endanger cells life cycle. For specific detection of CPDs the method of alkaline filter elution was adapted using T4 endonuclease V tratment of DNA. This enzyme introduces single strand breaks in the DNA at the CPD sites. The UV dose response was detected in two different cultures growing under 24/0 and 16/8 light/dark regime. Both cultures were exposed to several doses of artificial UV light, however, culture with 16/8 light regime showed higher sensitivity. Smaller amounts of UV absorbing and photosynthetic pigments were measured in respect to the 24/0 culture. Thus,pigments act as cell protecting elements and as UV target as well. For the same UV doses, removal of CPDs ( up to 24 hours of repair) was investigated. Depending on the dose , in one day 80-90% of CPD was repaired, but during the repair time phytoplankton cells did not duplicate and the normal cell dividing and culture growth was altered. Phytoplankton cultures were exposed to the sunlight in Rovinj at noon in midsummer for 3 hours. The amount of CPDs decreased between 1 hour and 3 hours suggesting that sunlight exposure induces not only DNA damage but also repair; both DNA damaging and repair are simultaneous. At present knowledge the potential damage of UV to phytoplankton populations can only be roughly estimated but our results showed that the consequences of increasing UV irradiation through weakening phytoplankton population could affect food chains in marine community.

U ovom radu istražen je učinak benzo(a)pirena (B(a)P) na integritet DNA u škrgama dagnje Mytilus galloprovincialis pomoću Fast mikrometode. Ta metoda detektira smanjenje integriteta DNA uslijed oštećenja pomoću fluorokromatskog bojila PicoGreen koji ima sposobnost vezanja za dvostruku DNA u alkalnim uvjetima. Jedinke dagnje su izlagane različitim koncentracijama B(a)P-a u vodi nakon čega je utvrđen dozni odgovor ukazujući na genotoksični učinak B(a)P-a u škrgama dagnje. Koncentracije B(a)P-a u vodi veće od 0.1 μg/L rezultiraju statistički značajnim smanjenjem integriteta DNA u škrgama. In this work, the effect of benzo (a) pyrene (B (a) P) on DNA integrity in the gills of the mussel Mytilus galloprovincialis was investigated using the Fast micromethod. This method detects a decrease in DNA integrity due to damage using the fluorochromatic dye PicoGreen which has the ability to bind to double DNA under alkaline conditions. Mussel specimens were exposed to different concentrations of B (a) P in seawater after which a dose response was determined indicating the genotoxic effect of B (a) P in mussel gills. Concentrations of B (a) P in water greater than 0.1 μg / L result in a statistically significant reduction in DNA integrity in gills.