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Silver nanoparticles represent a threat to biota and have been shown to cause harm through a number of mechanisms, using a wide range of bioassay endpoints. While nanoparticle concentration has been primarily considered, comparison of studies that have used differently sized nanoparticles indicate that nanoparticle diameter may be an important factor that impacts negative outcomes. In considering this, the aim of the present study was to determine if different sizes of silver nanoparticles (AgNPs; 10, 20, 40, 60 and 100 nm) give rise to similar effects during embryogenesis of Mediterranean sea urchins

Transplantation of mussels to new aquaculture sites is currently being undertaken in Istria (Croatia) to increase production, minimize potential seasonal urban contamination, and secure safer seafood products. The purpose of this study was to determine the effects of season and transplantation on physiological indices (condition index and meat yield) of mussels, Mytilus galloprovincialis. Mussels were obtained from five aquaculture areas in Istria (central Lim Bay, Rasa, Budava, Vabriga, and Pomer) and moved to a reference site in inner Lim Bay from April to July. Results showed that both season and transplantation affected physiological indices of mussels. Higher physiological indices were found in cultivated mussels from all areas in summer compared to spring, except in mussels from Vabriga and Pomer. In addition, higher physiological indices were found in mussels transplanted from central Lim Bay to the reference site in inner Lim Bay. Transplantation negatively affected physiological indices of mussels from Vabriga and Pomer, with no change in mussels from Rasa and Budava. In conclusion, mussels from all areas exhibit satisfactory commercial results (≥15%) during the peak summer harvesting season. For most transplanted mussels, transplantation itself represents additional stress, bringing into focus adaptation processes, stock genetics, and favorable ecological conditions.

The transfer of mariculture bivalves into new areas is increasing the risk of spreading Mytilicola copepods in native bivalve populations. Mytilicola orientalis was spread from Japan to native oysters and mussels in the Mediterranean. However, the presence of M. orientalis and its effect on the Mediterranean mussel, Mytilus galloprovincialis, are so far under-investigated. Our study shows the first identification of M. orientalis in mussels from the northern Adriatic Sea. M. orientalis was detected in mussels, from the Lim Bay, around the mariculture area of M. galloprovincialis. The adult stages of the copepod occur in mussels from May to August. The mean intensity of infestation and prevalence reached the maximum in August and was positively related to seawater temperature and salinity. We found one to three parasites per host individual, and most of the infested mussels were infested by one single parasite. Using the condition index, we found a relation between mussel physiology and seasonal cycle. Number of parasites was not related to the applied condition index. Our study established no difference in M. galloprovincialis condition index between infested and uninfested individuals, suggesting that the presence of M. orientalis does not adversely affect the mussel physiology to a measurable extent. However, our study reports the first record in the Adriatic Sea for M. orientalis.

The esterase activity of carbonic anhydrase (CA) was investigated in mussels sampled at 24 locations along the Croatian coast of Adriatic Sea. The gills were the target tissue because the respiratory, ionic transport and pH regulatory enzyme function of CA and its potential usage as biomarkers of environmental pollution was the main topic. Total esterase activity was measured in cytosolic fraction by colorimetric end-point reaction using p-nitrophenyl acetate as enzyme substrate. CA activity was estimated by the same enzymatic reaction using acetazolamide as a specific CA inhibitor. The results of total esterase activities in winter (March; all sites average value 0.137±0.057) were lower than determined for summer season (August; 0.153±0.036) at almost all investigated locations. CA activities determined in gills of mussel sampled in winter ranged from 1.75% to 24.65% of total esterase activities and in summer samples were lower (0.83 to 13.45%). Although recent research showed potential application of CA activity in bioassay and biomarker in pollution studies, further research is needed. Here we report a short-term simple colorimetric microplate method which can be applied for analyses of large numbers of samples. Furthermore in this study we characterized full length coding sequence (cDNA) of M. galloprovincialis carbonic anhydrase II (CAII). The CAII cDNA (with the 5’ and 3’ untranslated regions) is 1317 bp long. The putative open reading frame encodes a polypeptide of 256 amino acids, with a theoretical pI/Mw 5.87/28.416 kDa and conserved domains (active site and zinc binding site).

WOS: 000497259100061, PubMed: 31629182, Two microplastic sets, polystyrene (PS) and polymethyl methacrylate (PMMA), were tested for adverse effects on early life stages of Sphaerechinus granularis sea urchins. Microparticulate PS (10, 80 and 230 mu m diameter) and PMMA (10 and 50 mu m diameter) were tested on developing S. granularis embryos from 10 min post-fertilisation (p-f) to the pluteus larval stage (72 h p-f), at concentrations ranging from 0.1 to 5 mg L-1. Both PS and PMMA exposures resulted in significant concentration-related increase of developmental defects and of microplastic uptake in plutei. Moreover, embryo exposures to PS and PMMA (5 and 50 mg L-1) from 10 min to 5 h p-f resulted in a significant increase of cytogenetic abnormalities, expressed as significantly increased mitotic aberrations, while mitotoxicity (as % embryos lacking active mitoses) was observed in embryos exposed to PS, though not to PMMA. When S. granularis sperm suspensions were exposed for 10 min to PS or to PMMA (0.1-5 mg L-1), a significant decrease of fertilisation success was observed following sperm exposure to 0.1 mg L-1 PS, though not to higher PS concentrations nor to PMMA. Sperm pretreatment, however, resulted in significant offspring damage, as excess developmental defects in plutei, both following sperm exposure to PS and PMMA, thus suggesting transmissible damage from sperm pronuclei to the offspring. the overall results point to relevant developmental, cytogenetic and genotoxic effects of PS and PMMA microplastics to S. granularis early life stages, warranting further investigations of other microplastics and other target biota., Croatian Science Foundation [IP-2018-01-5351]; Environment and Climate Change Canada, This work has been supported by the Croatian Science Foundation under project IP-2018-01-5351. Financial support from Environment and Climate Change Canada is also gratefully acknowledged. Declarations of conflict: none.

The environmental status of the marine environment in the NE Adriatic Sea was assessed, using as a bioindicator species the Mediterranean mussel Mytilus galloprovincialis Lamarck, 1819. Samples were collected seasonally from mariculture sites and from major Istrian ports between the years 2010 and 2013. The condition indices of mussels ranged from 13.3 to 20.9% at mariculture sites and from 14.3 to 23.3% at port locations. The seasonally δ13CDIC values of seawater varied between -10.9 to 0.7‰. Pollution by sewage sludge (based on δ15N values) was confirmed only in two ports. Tissue concentrations of Mn, Co, Ni, Cu, Zn, Se, Cd, and Pb were significantly higher in the tissue of the mussels collected from the ports (polluted sites). Arsenobetaine was the major As compound present in the samples and there was no significant difference in the levels of total As in mussel tissues from mariculture and port sites.

Mussels Mytilus spp. belong to a group of key species in marine coastal ecosystems. This work demonstrated the dominant presence of Mediterranean mussel M. galloprovincialis Lmk. 1819 along the eastern Adriatic coast, from Lim bay to Dubrovnik. Morphological variations based on mussel shell measurements were within normal values for M. galloprovincials (h/l ratio 55.89 ± 5.08%) with several morphotypes determined by outline analysis. However, molecular analyses gave new insight into the genetic diversity of Adriatic mussel M. galloprovincialis. Previously, with the application of nuclear marker Me 15/16 analysis that distinguishes mussels of Mytilus edulis complex species (M. edulis-EE, M. trossulus-TT and M. galloprovincialis-GG), among mussels from all 22 sites, only 2 of 110 specimens were identified as heterozygote GE and GT genotypes, while all others were identified as homozygote GG. The amplified fragment length polymorphism (AFLP) analysis using our new nuclear precollagen-D gene marker (preCol-D) showed that the most prevalent Adriatic M. galloprovincialis preCol–D fragment size is 239/240 bp. Comparing the AFLP results of Adriatic M. galloprovincialis populations with Mytilus spp. (GG, GE, EE, ET and TT) from other locations (Mediterranean, Atlantic, Baltic, southern Pacific) it was possible to distinguish Adriatic-Mediterranean (239/240) and Atlantic (236/237 bp) M. galloprovincialis mussel populations as well as to identify all other species of M. edulis complex.

Statement of the Problem: Mytilicola orientalis is a co-invading copepod that have been co- introduced with infested oysters and mussels from Japan into North America, established self-sustaining populations and then spread from European Atlantic coast, to new, native hosts in Mediterranean (Francisco et al. 2010 ; Elsner et al. 2011 ; Pogoda et al. 2012). Relatively recent introduction of M. orientalis in the Mediterranean is of a great concern because it can spread and infest native mussel Mytillus galloprovincialis that are important aquaculture resources in Croatia (Pavičić-Hamer et al. 2016). The magnitude of the threat posed to native species by co-invaders will depend, among other things, on parasite ability to infect a native host (Lymbery et al. 2014). However, the presence and infection level of M. orientalis in native and farmed mussel from Adriatic Sea has not been previously studied. The purpose of this study is to observe the presence of M. orientalis in native M. galloprovincialis from Rovinj coastal area and aquaculture area in Lim Bay, the Northern Adriatic, Croatia. Methodology & Theoretical Orientation: Prevalence and intensity of infection with M. orientalis was observed from April to August 2012. Condition index was measured to understand the relationship between mussel physiological state and level of infection in which M. orientalis occurs. Findings: Our study shows the first identification of M. orientalis in the Northern Adriatic. The adult stages of the copepod occur in mussels from Lim Bay from May to August. The mean intensity of infestation and prevalence reached the maximum in August. Although, condition index was related with seasonal cycle, level of infection has no issue. Conclusion & Significance: Since we found copepod in mussels from nearby aquaculture area we suggest the transplantation of mussel seed in aquaculture area or/and co-introduction with non-native species could affect the spreading of M. orientalis in the new range.

The ability of mussels to cope with environmental conditions depends on the species’ capacity to acclimate or adapt within its morphometric and physiological parameters. In the present study,we investigated the effect of aquaculture site and translocation on different stocks of Mediterranean mussel, Mytilus galloprovincialis Lamarck, 1819. We used shell morphological ratios (width/length, height/length and height/width) and allometric relationships (volume-length and shell weight-length) in addition to condition index (wet meat weight/total weight) and mortality to compare cultivated and translocated mussels from the northern Adriatic Sea, Croatia. Mussels were collected from five cultivation areas (Lim, Pomer, Raša, Vabriga and Budava), and then moved for three months to experimental station at marine protected area in Lim Bay. Differences between cultivated mussels suggested morphological plasticity, although translocation weakly affected allometric relationships. Decrease in condition index was pronounced in translocated mussels, indicating adaptation to different local environmental conditions. High post translocation mortality was observed in mussels transferred to the experimental area. Our study shows plastic response of M. galloprovincialis, which can provide a useful information of mussel stocks selection in aquaculture.

Anthropogenic radioactivity in the northern Adriatic Sea was assessed by analyzing 137Cs activity concentrations in seawater, sediment, and marine organisms in the period from 2006 to 2010. The investigated locations were the Po River delta, Lim Bay and the Rovinj coastal area. At all locations 137Cs activity concentrations in seawater were lower than pre-Chernobyl values (< 4 Bq m-3). The highest 137Cs activity concentrations in sediment were found in the Po River delta (8.70 Bq kg-1). The data indicate that some species, such as sardine Sardina pilchardus, red mullet Mullus barbatus and the brown alga Fucus virsoides are better bioaccumulators of 137Cs than others. In addition, the ERICA Assessment Tool was used to calculate the total dose rates for reference organisms included in the software, as well as for specific sampled marine organisms.

We have assessed the activity of Na(+)/K(+)-ATPase, cAMP, free fatty acids (FFA) and metallothionein (MT) in the posterior gills of the brackish water shore crab Carcinus aestuarii during acclimation to 10 ppt dilute seawater (DSW). Following 3-18 days acclimation in DSW specific activity of Na(+)/K(+)-ATPase in native gill homogenates and partially purified membrane vesicles was progressively increased, from 1.7- to 3.9-fold. After short-term acclimation of crabs in DSW with added sucrose to make media isosmotic with the haemolymph the specific Na(+)/K(+)-ATPase activity in homogenates was not increased, relative to SW enzyme activity. Moreover, hyposmotic conditions led to depletion of cAMP in gills. In partially purified membrane vesicles isolated from posterior gills, fatty acids with compositions 16:0, 18:0, 18:1, 20:4 and 20:5 dominated in both SW- and DSW-acclimated Carcinus. During a year in which the metabolic activity of crabs was increased, the arachidonic/linoleic acids ratio (ARA/LA) for DSW-acclimated crabs was markedly increased relative to that in SW. Increased Na(+)+K(+)-ATPase activity under hyposmotic stress may be modulated at least partially by the changed proportion of fatty acids in the purified membranes of posterior gills. Long-term acclimation of shore crabs to DSW resulted in a 2.6-fold increase in cytosolic metallothionein (MT) content in posterior gills over those in SW crabs. Assuming an antioxidant role of MT associated with intracellular zinc partitioning, the observed MT induction in posterior gills may be considered an adaptive response of C. aestuarii to hyposmotic stress.

Total arsenic concentration in the edible part of mussels Mytilus galloprovincialis was evaluated seasonally in the coastal area of Rijeka Bay (North Adriatic Sea, Croatia). Sampling stations were located close to the City of Bakar with no industrial facilities (site 1), in the vicinity of the oil refinery and oil thermoelectric power plant (Urinj, site 2), and 4 miles away from the Plomin coal thermoelectric power plant (Brsec village, site 3). Additionally, the concentration of arsenic in the tail muscle of the lobster Nephrops norvegicus, collected in Rijeka Bay, was studied. During winter at sites 2 and 3, the total arsenic in the edible part of the mussels was 16.4 mg As/kg FW (FW=fresh weight) and 4.38 mg As/kg FW, respectively, and increased during springtime at site 2 (6.5 mg As/kg FW) compared to the rest of the year, when individual total arsenic concentration at all sites ranged from 1.7 to 3.7 mg As/kg FW. In the winter (sites 2 and 3) and springtime (site 2) there was no correlation between the length of the mussel shell and the arsenic concentration in the edible part of the mussels. In the other seasons, at sites 1, 2 and 3, there was a correlation between arsenic in the edible part of mussels and shell length in most cases (correlation coefficients r varied from 0.64 to 0.85; P

Activities of carbonic anhydrase (CA) and its distribution in the branchial cavity tissues were studied in European lobsters ( Homarus gammarus ) from ambient seawater (SW; salinity=38 ppt, 1126 mosmol/l) and acclimated to dilute seawater (DSW; salinity=20 ppt, 548 mosmol/l). Acetazolamide inhibited dose dependently the activity of CA in homogenates of epipodites, where the inhibition constant (IC 50 =0.12 μM) did not differ significantly from that of membrane vesicles and cytosolic fraction. In DSW-acclimated lobsters, almost 70% of total CA in tissues of the branchial cavity was found in epipodites (E) and the rest was equally distributed between branchiostegites (B) and gills (G). Upon acclimation to dilute seawater, CA activity in membrane fractions of E and B was increased 6-fold and in homogenates, respectively 5- and 13-fold compared to SW-acclimated lobsters. Exposure to DSW enhanced cytosolic CA in E (8-fold) and B (7-fold) over SW-acclimated animals. Slight activation of CA in homogenates and in partially purified membranes of G was not confirmed as a statistically significant difference between SW and DSW groups. In DSW, cytosol specific activity of CA was increased compare to the SW cytosol. These results indicate the importance of E and B in CA induction when lobsters are acclimated to DSW. In subcellular fractions from DSW-acclimated lobsters, the main proportion of 75.8% (E), 61.0% (B) of total CA activity in each of these tissues remained in cytosol portion. Partially purified membranes contained 6.8% (E) and 16.2% (B) and the remainder of 15% (E) and 27% (B) was found in mitochondrial and nuclear fractions. In gills, 49.2% and 9.0 % of total gill CA activity was found respectively in cytosol and partially purified vesicles and the rest in mitochondrial and nuclear fractions.

Artificial and natural radionuclides were determined in the Adriatic Sea in the seawater and sediment samples in the period from 2007 to 2011. The sampling areas were coastal waters of Slovenia, Croatia and Albania, together with the deepest part of the Adriatic in South Adriatic Pit and Otranto strait. Sampling locations were chosen to take into account all major geological and geographical features of this part of the Adriatic Sea and possible coastal influences. After initial sample preparation steps, samples were measured by gamma-ray spectrometry. In the seawater 40K activity concentrations were in the range from 6063 to 10519 Bq m23, 137Cs from 1.6 to 3.8 Bq m23, 226Ra from 23 to 31 Bq m23, 228Ra from 1 to 25 Bq m23 and 238U from 64 to 490 Bq m23. The results of sediment samples showed that 40K was in the range from 87 to 593 Bq kg21, 137Cs from 0.8 to 7.3 Bq kg21, 226Ra from 18 to 35 Bq kg21, 228Ra from 4 to 29 Bq kg21 and 238U from 14 to 120 Bq kg21. In addition, the ERICA Assessment Tool was used for the assessment of dose rates for reference marine organisms using the activity concentrations of the determined radionuclides in seawater. The assessment showed that for the most of the organisms, the dose rates were within the background levels, indicating that the determined values for seawater does not pose a significant risk for the most of marine biota. In the study, the results are critically discussed and compared with other similar studies worldwide. Generally, the activity concentrations of the examined radionuclides did not differ from those reported for the rest of the Mediterranean Sea.

In the present study, we investigated the progressive acclimation of the mussel Mytilus galloprovincialis to different reduced seawater (SW) salinities and its effect on several biochemical markers and biotests. Mussels were purchased from a local mariculture facility during summer (SW temperature 27 degrees C, salinity 37.5 psu) and winter (13 degrees C, 37 psu) seasons, and transferred to the laboratory for acclimation to reduced SW salinities (37, 28, 18.5 and 11 psu). At the beginning and at the end of acclimation processes tests of mussel survival in air were provided. After 14 days of acclimation the DNA integrity, p38-MAPK activation, metallothionein induction, oxygen consumption rate, and condition index were measured. Survival in air (SOS test), as a physiological index of mussel's health and vitality, had significantly lower LT50 values (11 psu) in the summer than in the winter, and it seems to be negatively affected by acclimation in comparison to controls (37 psu and mariculture). Condition indexes (CIs) were not significantly different, but mussel's acclimation resulted in decline (i.e., a negative trend), especially of CI-2 and CI-3 calculated on the basis of mussel tissue weight and shell sizes. Oxygen consumption rate (VO2) of M. galloprovincialis acclimated to reduced salinities was a concentration-dependent process and increased considerably to about 51 and 65% in lower SW concentrations (28 and 18 psu) compared to control mussels (37 psu). DNA integrity, determined by Fast Micromethod, was negatively impacted by salinity acclimation and corresponding physiological stress as well. Some differences in 1D protein expression patterns between control groups and mussels acclimated to 28, 18.5 and 11 psu (SW) were established. Reduced SW salinities (18.5 and 11 psu) resulted in significantly higher p38-MAPK phosphorylation, whereas the SW salinity of 28 psu decreased p-p38 significantly compared to control (37 psu). The concentration of metallothioneins in mussels' gills was reduced at 28 and 18.5 psu, while it was significantly higher at 11 psu. Results indicated that SW salinity variation (i.e., hypoosmotic stress) in the marine environment can affect all investigated parameters. This investigation expands our understanding of multifactorial effects of the physical marine environment on the specificity of investigated biomarkers and biotests, providing insight into the acclimation, adaptive and stress response processes of mussels. Effects of environmental factors have to be considered in sampling strategies for monitoring programmes to prevent false interpretation of results.