Will extreme weather events influence the toxic impacts of caffeine in coastal systems? Comparison between two widely used bioindicator species.

In the recent years, marine heatwaves (MHWs) have caused devastating impacts on marine life. The understanding of the combined effects of these extreme events and anthropogenic pollution is a vital challenge. In particular, the combined effect of MHWs on the toxicity of pharmaceuticals to aquatic life remains unclear. To contribute to these issues, the main goal of the present investigation was to evaluate how MHWs may increase caffeine (CAF) toxicity on the clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis. Bioaccumulation levels and changes on oxidative stress, metabolic capacity and neurotoxic status related biomarkers were investigated. The obtained results revealed the absence of CAF accumulation in both species. However, the used contaminant generated in both bivalve species alteration on neurotransmission, detoxification mechanisms induction as well as cellular damage. The increase of antioxidant defence mechanisms was complemented by an increase of metabolic activity and decrease of energy reserves. The obtained results seemed magnified under a simulated MHWs, suggesting to a climate-induced toxicant sensitivities' response. On this perspective, understanding of how toxicological mechanisms interact with climate-induced stressors will provide a solid platform to improve effect assessments for both humans and wildlife.

Bioassay-based ecotoxicological investigation on marine and freshwater impact of cigarette butt littering.

Despite representing an extremely relevant portion (20-40%) of worldwide coastal litter, cigarette butts are still an underestimate environmental issue of limited scientific interest. Public authorities of different countries promote active removal of cigarette butts, but the issue remains problematic in terms of aesthetic, environmental and health-related impacts. There are few studies on the environmental side-effects of smoked cigarette butt litter despite being a worldwide issue. In this work, two ecotoxicological bioassay batteries were adopted to evaluate the environmental consequences of cigarette butt water-soluble ingredient release in both marine water and freshwater. Marine assays were generally more affected compared to freshwater. Interesting outcomes were observed with crustacean tests, showing a lower effect of smoked cigarette butt leachate when tested at maximum concentration. This finding were supported by heartbeat measures of Daphnia magna, which were accelerated at 100% of smoked cigarette butt leachate.

Investigation of mechanisms underlying chaotic genetic patchiness in the intertidal marbled crab Pachygrapsus marmoratus (Brachyura: Grapsidae) across the Ligurian Sea.

BACKGROUND: Studies on marine community dynamics and population structures are limited by the lack of exhaustive knowledge on the larval dispersal component of connectivity. Genetic data represents a powerful tool in understanding such processes in the marine realm. When dealing with dispersion and connectivity in marine ecosystems, many evidences show patterns of genetic structure that cannot be explained by any clear geographic trend and may show temporal instability. This scenario is usually referred to as chaotic genetic patchiness, whose driving mechanisms are recognized to be selection, temporal shifts in local population dynamics, sweepstakes reproductive success and collective dispersal. In this study we focused on the marbled crab Pachygrapsus marmoratus that inhabits the rocky shores of the Mediterranean Sea, Black Sea and East Atlantic Ocean, and disperses through planktonic larvae for about 1 month. P. marmoratus exhibits unexpectedly low connectivity levels at local scale, although well-defined phylogeographic patterns across the species' distribution range were described. This has been explained as an effect of subtle geographic barriers or due to sweepstake reproductive success. In order to verify a chaotic genetic patchiness scenario, and to explore mechanisms underlying it, we planned our investigation within the Ligurian Sea, an isolated basin of the western Mediterranean Sea, and we genotyped 321 individuals at 11 microsatellite loci. RESULTS: We recorded genetic heterogeneity among our Ligurian Sea samples with the occurrence of genetic clusters not matching the original populations and a slight inter-population divergence, with the geographically most distant populations being the genetically most similar ones. Moreover, individuals from each site were assigned to all the genetic clusters. We also recorded evidences of self-recruitment and a higher than expected within-site kinship. CONCLUSIONS: Overall, our results suggest that the chaotic genetic patchiness we found in P. marmoratus Ligurian Sea populations is the result of a combination of differences in reproductive success, en masse larval dispersion and local larval retention. This study defines P. marmoratus as an example of marine spawner whose genetic pool is not homogenous at population level, but rather split in a chaotic mosaic of slightly differentiated genetic patches derived from complex and dynamic ecological processes.

Atlantic and Mediterranean populations of the widespread serpulid Ficopomatus enigmaticus: Developmental responses to carbon nanotubes.

Ficopomatus enigmaticus was adopted as model species for ecotoxicological bioassay, with its larval development as endpoint. Two different populations of the same species, collected in areas far from each other (Mediterranean Sea and Atlantic Ocean), were exposed to multi-walled carbon nanotubes, a class of emerging pollutants with a constantly increasing relevance in the landscape of nanomaterials production. Moreover, a molecular analysis based on Cyt b amplification and sequencing, was carried out to confirm that both populations belong to the same species. The aim of the present work was to strengthen existing results about F. enigmaticus relevance in ecotoxicological bioassays, adding the variable of population effect. For both populations the concentration-response curve of effect at different toxicant concentrations was similar and, at certain concentrations, overlapping, confirming the ecological relevance of the assay. These results posed an interesting acceptance on the introduction of this species as model in ecotoxicological bioassay scenery, underlining the relevance of a widespread wild species to compare effects of chemicals and environmental samples over large distances using the same bioassay.

Effects of copper on larvae of the marbled crab Pachygrapsus marmoratus (Decapoda, Grapsidae): Toxicity test and biochemical marker responses.

The importance of trace elements in ecotoxicological investigations is a well-known issue when monitoring polluted areas such as commercial harbors. Copper represents one of the most common metal contaminants, often detected in these areas as it is widely employed in various fields and has many sources of inflow in the marine environment. Pachygrapsus marmoratus is a widespread intertidal crab species that has been extensively studied in ecology, ethology and population genetics. Ecotoxicological studies have also been performed, exclusively on the adult stage. In the present study we investigated the mortality and biochemical (oxidative stress and neurotoxicity) responses of P. marmoratus larvae exposure to environmental relevant concentration of copper. Results showed dose-dependent responses in terms of larval mortality, with a calculated LC50 value of 0.5 mg/L of Cu2+. The LC50 concentration was used as the starting point for subsequent biochemical response evaluation. Results also demonstrated dose-dependent activation of antioxidant systems assuming a compensatory antioxidant activity to prevent higher cellular damage when larvae were exposed to the highest concentrations of copper. Moreover, a significant enhancement of neurotransmitter activities was observed, assuming a possible direct interaction of copper with the enzymes or an increase of free copper ion aliquot into the cells.

Ficopomatus enigmaticus larval development assay: An application for toxicity assessment of marine sediments.

Ficopomatus enigmaticus is a serpulid polychaete, reef-forming, dioecious gamete spawner throughout different seasons and in different salinity conditions. Due to these characteristics, its candidacy as a model organism to monitor both marine and brackish matrices was assessed. Marine sediments collected in a polluted area near the port of Ancona (Italy) were used as case study. Besides the chemical characterization of sediments, the larval development of F. enigmaticus was adopted as an assay for ecotoxicological assessment of sediments in addition to a regulatory bioassay battery with different organisms and endpoints. The quality of sediments was classified according to the Italian Ministerial Decree for sediment management (Ministerial Decree 173, 2016) by using the SEDI-QUAL-SOFT® (ver. 109.0) software developed by the Italian Environmental Protection Agency (ISPRA). The F. enigmaticus larval development assay showed a quite similar sensitivity to C. gigas larval development assays, indicating its potential use in a regulatory battery of bioassays.

Antimicrobial susceptibility of Staphylococcus intermedius and Staphylococcus schleiferi isolated from dogs.

The susceptibility to 23 antimicrobial agents was determined in 114 isolates of Staphylococcus intermedius and eight isolates of Staphylococcus schleiferi of canine origin. Overall, 73% of S. intermedius isolates and 37.5% of S. schleiferi isolates were susceptible to all the 23 antimicrobials tested. The large majority of S. intermedius strains retained susceptibility to antimicrobials currently employed in treatment of pyoderma (cephalosporins, cotrimoxazole and association amoxicillin-clavulanic acid) as well as to those effective against staphylococci (fusidic acid, rifampicin and fluoroquinolones). Resistance in S. intermedius was observed mainly against macrolides, chloramphenicol and lincosamides, while S. schleiferi isolates retained susceptibility to all antimicrobials except three of six fluoroquinolones. Although, our results confirm susceptibility to antimicrobials currently employed in pyoderma treatment, the several different resistance patterns observed for S. intermedius emphasize the importance of antimicrobial susceptibility testing of canine staphylococci to choose the most appropriate treatment of infections and to allow the prudent use of antimicrobial drugs in companion animals.

An optimized digestion method coupled to electrochemical sensor for the determination of Cd, Cu, Pb and Hg in fish by square wave anodic stripping voltammetry.

An optimized digestion method coupled to electrochemical detection to monitor lead, copper, cadmium and mercury in fish tissues was developed. Square wave anodic stripping voltammetry (SWASV) coupled to disposable screen-printed electrodes (SPEs) was employed as fast and sensitive electroanalytical method for heavy metals detection. Different approaches in digestion protocols were assessed. The study was focused on Atlantic hake fillets because of their wide diffusion in the human nutrition. Best results were obtained by digesting fish tissue with hydrogen peroxide/hydrochloric acid mixture coupled to solid phase (SP) purification of the digested material. This combined treatment allowed quantitative extraction from fish tissue (muscle) of the target analytes, with fast execution times, high sensitivity and avoiding organic residues eventually affecting electrochemical measurements. Finally, the method has been validated with reference standard materials such as dogfish muscle (DORM-2) and mussel tissues (NIST 2977).

Antimicrobial susceptibility and mechanism of resistance to fluoroquinolones in Staphylococcus intermedius and Staphylococcus schleiferi.

The objective of the present study was to determine the antimicrobial susceptibility of 136 canine isolates of Staphylococcus intermedius and 10 canine isolates of S. schleiferi subspecies coagulans to 16 fluoroquinolones (FQs), and to investigate the mechanisms of resistance in the nonsusceptible isolates. Of the 136 of S. intermedius tested 98.5% were susceptible to all 16 FQs whereas only 40% of the 10 isolates of S. schleiferi subspecies coagulans were susceptible. Two isolates of S. intermedius and six isolates of S. schleiferi, were found to be resistant to 13 out of 16 FQs, while they retained their susceptibility to fourth generation FQs such as gatifloxacin, moxifloxacin and trovafloxacin. Sequencing of the quinolone-resistance determining regions of gyrA and grlA genes showed that in S. intermedius, dichotomous resistance to FQs was associated with the occurrence of one alteration in GyrA-84 and one in GrlA-80, while in S. schleiferi the same pattern of resistance was observed in isolates showing these changes only in gyrA. This study is the first to screen FQs of the second, third and fourth generation for antimicrobial resistance in clinical isolates of S. intermedius and S. schleiferi of canine origin, and to describe mutations in gyrA and grlA associated with FQ resistance in these bacterial species.

An optical immunosensor for rapid vitellogenin detection in plasma from carp (Cyprinus carpio).

Vitellogenin (vtg) has proven to be a sensitive and simple biomarker for assessing exposure of fish to environmental estrogens. The aim of this work was to develop a rapid, in the order of minutes, screening method for the detection of fish vtg. The surface plasmon resonance technique (Biacore Xtrade mark) was coupled with immunodetection for the determination of fish vtg in plasma and mucus from carp (Cyprinus carpio). Monoclonal anti-vtg antibodies were linked on the sensor surface through chemical cross-linking via a capturing antibody. A simple regeneration process allowed the reuse of the sensor surface. Sensor optimisation was carried out using carp vtg. The developed immunosensor was tested with vtg spiked samples and with plasma and mucus from fish exposed to 17beta-estradiol (E2). Vitellogenin could be detected in the ppm range in buffer as well as in plasma and mucus. Good discrimination between control and exposed samples was obtained. The results were compared with ELISA and a correlation coefficient of R(2)=0.85 (n=9) between the two methods indicated that the immunochemical biosensor could be used for the analysis of vtg in fish plasma samples. The assay time was 20min hence allowing for rapid sample screening.

Safety of azamethiphos in eel, seabass and trout.

The safety of azamethiphos (AZA), an organophosphorous insecticide and the active ingredient of Salmosan, was evaluated in the European eel, seabass and rainbow trout. Fish were bathed in 0.1 ppm AZA for a period of 60, 120 or 240 min. After termination of each treatment fish were transferred to clean aquaria and randomly sampled over 21 days. Compared to controls, brain acetylcholinesterase (AChE) was inhibited up to 44, 56 and 62% in eels, seabass and trout, respectively, with the inhibition being significant for up to 4 days in eels and seabass and 7 days in trout. As result of the AChE depression, fish displayed motor hyperactivity and erratic jumping at the onset of treatment. Mortality was observed only in trout following exposure for 240 min. A variable correlation observed among species between the level of exposure, the reduced activity of brain AChE and the signs of toxicity suggest that brain AChE should be considered as an indicator of exposure rather than as an index of toxicity of AZA. The present data indicate that at the therapeutic dosage of 0.1 ppm AZA for 1h can be safely used in eels, seabass and trout. The extended treatment times up to 240 min were equally safe for eels and seabass but not for trout.

Effects of beta-naphthoflavone on the cytochrome P450 system, and phase II enzymes in gilthead seabream (Sparus aurata).

The effect of beta-naphthoflavone (beta-NF) on several catalytic activities of cytochrome P450 (CYP) and phase II enzymes putatively controlled by [Ah]-receptor activation in the liver, heart and kidney of gilthead seabream, was investigated. In the liver, beta-NF treatment [intraperitoneal injection (i.p.) 50 mg/kg] resulted in an increase of CYP content, immunoreactive CYP 1A and methoxyresorufin-O-demethylase (MEROD), pentoxyresorufin O-depentylase (PROD) and ethoxyresorufin-O-deethylase (EROD) activities. However, beta-NF had no effect on any of the hepatic phase II enzymes examined (benzaldehyde dehydrogenase, propionaldehyde dehydrogenase, glutathione S-transferase, UDP-glucuronyl-transferase, DT-diaphorase). Single i.p. injection of 10 mg/kg beta-NF showed a maximal induction of CYP 1A-like protein and EROD activity after 3-7 days. CYP 1A and EROD returned to control levels 18-days post-treatment. beta-NF injection also caused a rapid increase of a single band size of mRNA recognized by a CYP 1A1 cDNA fragment from sea bass (Dicentrarchus labrax). Expression of mRNA preceded the increase of EROD activity and declined rapidly by 96 h. Dose-response experiments demonstrated that EROD was significantly enhanced in liver by a single injection of 0.3 mg/kg beta-NF and was the most sensitive measurement for CYP 1A-like induction. beta-NF treatments also increased the expression of CYP 1A-like protein, mRNA and EROD, but not MEROD and PROD activities in heart and kidney.

Effect of clofibrate, a peroxisome proliferator, in sea bass (Dicentrarchus labrax), a marine fish.

The effects of 35 or 70 mg/kg clofibrate on some peroxisomal, mitochondrial, and microsomal enzymes (markers of peroxisome proliferation) in liver, gill, and kidney of sea bass following 2 weeks i.p. treatment have been studied. Induction of lauric acid hydroxylase, UDP-glucuronyl transferase, palmitoyl-CoA oxidase, carnitine-palmitoyl-transferase, p-nitrophenylacetate hydrolase, and benzaldehyde and propionaldehyde dehydrogenase activities was not observed in any case. The clofibrate administration at the dose of 70 mg/kg induced in the liver the glutathione S-tranferase and reduced epoxide hydrolase activities. These results demonstrate that a marine fish species, such as sea bass, is refractory to peroxisome proliferation.