Cell cycle alterations due to perfluoroalkyl substances PFOS, PFOA, PFBS, PFBA and the new PFAS C6O4 on bottlenose dolphin (Tursiops truncatus) skin cell.

Per- and polyfluoroalkyl substances (PFAS) have become ubiquitous environmental contaminants in aquatic ecosystems worldwide. Marine mammals, as top predators, are constantly exposed to several PFAS compounds that accumulate in different tissues. As a proxy to assess cytotoxicity of PFAS in the bottlenose dolphin (Tursiops truncatus), we generated a new immortalized cell line derived from skin samples of bottlenose dolphin. Using high content imaging, we assessed the effects of increasing concentrations of PFOS, PFOA, PFBS, PFBA and C6O4 on cell viability and cell cycle phases. In particular, we classified all cells based on multiple morphometric differences of the nucleus in three populations, named respectively "Normal" (nuclei in G0, S and M phase); "Large" (nuclei showing characteristics of senescence) and "Small" (nuclei with fragmentation and condensed chromatin). Combining this approach with cell cycle analysis we determined which phases of the cell cycle were influenced by PFAS. The results revealed that the presence of PFOS, PFBS and PFBA could increase the number of cells in G0+G1 phase and decrease the number of those in the S phase. Moreover, PFOS and PFBS lowered the fraction of cells in the M phase. Interestingly PFOS, PFBS and PFBA reduced the prevalence of the senescence phenotype ("large" nuclei), suggesting a potential tumorigenic effect. Besides, the presence of PFOS and PFBS correlated also with a significant decrease in the number of "small" nuclei. The C6O4 exposure did not highlighted morphometric alteration or cell cycle modification bottlenose dolphin skin cell nuclei. While the effects of PFAS on cell cycle was clear, no significant change was detected either in term of cell proliferation or of viability. This study fosters the overall knowledge on the cellular effects of perfluoroalkyl substances in marine mammals.

Dynamics of blood circulation during diving in the bottlenose dolphin (Tursiops truncatus): the role of the retia mirabilia.

The retia mirabilia are vascular nets composed of small vessels dispersed among numerous veins, allowing blood storage, regulation of flow and pressure damping effects. Here, we investigated their potential role during the diving phase of the bottlenose dolphin (Tursiops truncatus). To this effect, the whole vertebral retia mirabilia of a series of dolphins were removed during post-mortem analysis and examined to assess vessel diameters, and estimate vascular volume and flow rate. We formulated a new hemodynamic model to help clarify vascular dynamics throughout the diving phase, based on the total blood volume of a bottlenose dolphin, and using data available about the perfusion of the main organs and body systems. We computed the minimum blood perfusion necessary to the internal organs, and the stroke volume and cardiac output during the surface state. We then simulated breath-holding conditions and perfusion of the internal organs under the diving-induced bradycardia and reduction of stroke volume and cardiac output, using 10 beats min-1 as the limit for the heart rate for an extended dive of over 3 min. Within these simulated conditions, the retia mirabilia play a vital role as reservoirs of oxygenated blood that permit functional performances and survival of the heart and brain. Our theoretical model, based on the actual blood capacity of the retia mirabilia and available data on organ perfusion, considers the dynamic trend of vasoconstriction during the diving phase and may represent a baseline for future studies on the diving physiology of dolphins and especially for the blood supply to their brain.

Determination of perfluoroalkyl acids in different tissues of graminaceous plants.

A method for the determination of 12 perfluoroalkyl acids (PFAA) in vegetal samples was proposed. The analytical procedure was developed to optimize the detection of short-chain PFAA (C < 8) due to their higher potential to be translocated and bioaccumulated in plants than long-chain congeners. The method, based on ultrasonic extraction, clean-up and HPLC-MS/MS analysis, determined PFAA in different plant tissues allowing the PFAA distribution and partition in vegetal compartments to be studied. The performance of this analytical procedure was validated by analysing samples (root, stem and leaf) of reed grass. The validated method was then applied to graminaceous plants from an agricultural area impacted by a fluorochemical plant discharge (Northern Italy). The PFAA congeners were detected in most of the samples with ΣPFAA concentrations in the whole plant ranging from

Evaluation of per- and poly-fluorinated alkyl substances (PFAS) in livers of bottlenose dolphins (Tursiops truncatus) found stranded along the northern Adriatic Sea.

Per-and poly-fluorinated alkyl substances (PFAS) are a group of chemicals used in a wide variety of commercial products and industrial applications. These chemicals are persistent, can accumulate in humans' and animals' tissues and in the environment, representing an increasing concern due to their moderate to highly toxicity. Their global distribution, persistence and toxicity led to an urgent need to investigate bioaccumulation also in marine species. In 2013 PFAS contamination was detected in a vast area in Veneto region, mainly in Adige and Brenta rivers. In order to investigate any relevant presence of these substances in marine vertebrates constantly living in the area, PFAS were measured in hepatic tissue samples of 20 bottlenose dolphins (Tursiops truncatus) stranded along the northern Adriatic Sea coastline between 2008 and 2020. Using high performance liquid chromatography-mass spectrometry, 17 target PFAS (PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnA, PFDoA, PFTrDA, PFTeDA, PFBS, PFHxS, PFOS, PFDS, PFHpS, PFPeS), were quantified in the samples. PFAS profiles were generally composed of the same five dominant PFAS (PFOS > PFUnA > PFDA ≈ PFDoA ≈ PFTrDA). The greatest PFOS concentration found was 629,73 ng/g wet weight, and PFOS accounted until 71% in the PFAS profiles. No significant differences between sexes were found, while calves showing higher mean values than adults, possibly indicating an increasing ability in the elimination of PFAS with age. Finally, a temporal analysis was carried out considering three different periods of time, but no temporal differences in concentrations were found. The results suggest that long-chain PFAS are widespread in bottlenose dolphins along the North Adriatic Sea. Furthermore, they represent a baseline to investigate the impact of PFAS on marine mammals' conservation and health. Filling an important gap in the knowledge of PFAS accumulation in bottlenose dolphins, this study highlights the relevant role of Environmental and Tissue Banks for retrospective analyses on emergent contaminants.

Correction: The use of Unmanned Aerial Vehicles (UAVs) to sample the blow microbiome of small cetaceans.

[This corrects the article DOI: 10.1371/journal.pone.0235537.].

The new PFAS C6O4 and its effects on marine invertebrates: First evidence of transcriptional and microbiota changes in the Manila clam Ruditapes philippinarum.

There is growing concern for the wide use ofperfluorooctanoic acid (PFOA) because of its toxic effects on the environment and on human health. A new compound - the so called C6O4 (perfluoro ([5-methoxy-1,3-dioxolan-4-yl]oxy) acetic acid) - was recently introduced as one of the alternative to traditional PFOA, however this was done without any scientific evidence of the effects of C6O4 when dispersed into the environment. Recently, the Regional Agency for the Protection of the Environment of Veneto (Italy) detected high levels of C6O4 in groundwater and in the Po river, increasing the alarm for the potential effects of this chemical into the natural environment. The present study investigates for the first time the effects of C6O4 on the Manila clam Ruditapes philippinarum exposed to environmental realistic concentrations of C6O4 (0.1 µg/L and 1 µg/L) for 7 and 21 days. Furthermore, in order to better understand if C6O4 is a valid and less hazardous alternative to its substitute, microbial and transcriptomic alterations were also investigated in clams exposed to 1 µg/L ofPFOA. Results indicate that C6O4 may cause significant perturbations to the digestive gland microbiota, likely determining the impairment of host physiological homeostasis. Despite chemical analyses suggest a 5 times lower accumulation potential of C604 as compared to PFOA in clam soft tissues, transcriptional analyses reveal several alterations of gene expression profile. A large part of the altered pathways, including immune response, apoptosis regulation, nervous system development, lipid metabolism and cell membrane is the same in C6O4 and PFOA exposed clams. In addition, clams exposed to C6O4 showed dose-dependent responses as well as possible narcotic or neurotoxic effects and reduced activation of genes involved in xenobiotic metabolism. Overall, the present study suggests that the potential risks for marine organism following environmental contamination are not reduced by replacing PFOA with C6O4. In addition, the detection of both C6O4 and PFOA into tissues of clams inhabiting the Lagoon of Venice - where there are no point sources of either compounds - recommends a similar capacity to spread throughout the environment. These results prompt the urgent need to re-evaluate the use of C6O4 as it may represent not only an environmental hazard but also a potential risk for human health.

PFAS Environmental Pollution and Antioxidant Responses: An Overview of the Impact on Human Field.

Due to their unique properties, perfluorinated substances (PFAS) are widely used in multiple industrial and commercial applications, but they are toxic for animals, humans included. This review presents some available data on the PFAS environmental distribution in the world, and in particular in Europe and in the Veneto region of Italy, where it has become a serious problem for human health. The consumption of contaminated food and drinking water is considered one of the major source of exposure for humans. Worldwide epidemiological studies report the negative effects that PFAS have on human health, due to environmental pollution, including infertility, steroid hormone perturbation, thyroid, liver and kidney disorders, and metabolic disfunctions. In vitro and in vivo researches correlated PFAS exposure to oxidative stress effects (in mammals as well as in other vertebrates of human interest), produced by a PFAS-induced increase of reactive oxygen species formation. The cellular antioxidant defense system is activated by PFAS, but it is only partially able to avoid the oxidative damage to biomolecules.

Copper/Zinc Superoxide Dismutase from the Crocodile Icefish Chionodraco hamatus: Antioxidant Defense at Constant Sub-Zero Temperature.

In the present study, we describe the purification and molecular characterization of Cu,Zn superoxide dismutase (SOD) from Chionodraco hamatus, an Antarctic teleost widely distributed in many areas of the Ross Sea that plays a pivotal role in the Antarctic food chain. The primary sequence was obtained using biochemical and molecular biology approaches and compared with Cu,Zn SODs from other organisms. Multiple sequence alignment using the amino acid sequence revealed that Cu,Zn SOD showed considerable sequence similarity with its orthologues from various vertebrate species, but also some specific substitutions directly linked to cold adaptation. Phylogenetic analyses presented the monophyletic status of Antartic Teleostei among the Perciformes, confirming the erratic differentiation of these proteins and concurring with the theory of the "unclock-like" behavior of Cu,Zn SOD evolution. Expression of C. hamatus Cu,Zn SOD at both the mRNA and protein levels were analyzed in various tissues, highlighting the regulation of gene expression related to environmental stress conditions and also animal physiology. The data presented are the first on the antioxidant enzymes of a fish belonging to the Channichthyidae family and represent an important starting point in understanding the antioxidant systems of these organisms that are subject to constant risk of oxidative stress.

Uptake and translocation of perfluoroalkyl acids (PFAAs) in hydroponically grown red chicory (Cichorium intybus L.): Growth and developmental toxicity, comparison with growth in soil and bioavailability implications.

Short-chain perfluoroalkyl acids (PFAAs) have shown a high potential for plant (crop) uptake, making them possibly significant contributors to the total dietary exposure to PFAAs. The plant uptake of PFAAs is a complex process that needs better characterization, as it does not only depend on perfluoroalkyl chain length, but also on their polar terminal group, on the plant species and the exposure media. Here, a plant uptake study with nine perfluoroalkyl acids (PFAAs) was carried out under the hydroponic (soilless) exposure conditions. Red chicory was grown in a nutrient solution, spiked with PFAAs mixture at three different concentrations (i.e. 62.5, 125 and 250 µg/L), in order to extend the range of levels tested and reported in the literature so far. Bioaccumulation metrics and transpiration stream concentration factors (TSCFs) were employed for the plant uptake characterization and consequent comparison with the results of soil uptake experiment we previously performed with the same crop. The results showed that calculated root concentration factors (RCFs) increase with PFAA chain length, while the opposite chain length dependence was present for shoots. Plants from two treatments with the highest PFAAs concentrations manifested physiological changes (discoloration, inhibited roots and leaves growth), despite of the used exposure concentrations being much lower than previously published phytotoxicity thresholds. A comparison among RCFs and TSCFs derived from hydroponic and from the soil experiment has emphasized their different magnitudes and PFAAs chain length dependence patterns. They could not be ascribed only to soil sorption as a process decreasing PFAAs bioavailability for plants, but also to developmental differences between the root systems formed in soil and in nutrient solution and to the potential competitive PFAAs sorption to roots in hydroponics. The interchangeable use of bioaccumulation and translocation parameters derived in hydroponic and soil systems would lead to erroneous conclusions and plant uptake predictions.

The use of Unmanned Aerial Vehicles (UAVs) to sample the blow microbiome of small cetaceans.

Recent studies describe the use of UAVs in collecting blow samples from large whales to analyze the microbial and viral community in exhaled air. Unfortunately, attempts to collect blow from small cetaceans have not been successful due to their swimming and diving behavior. In order to overcome these limitations, in this study we investigated the application of a specific sampling tool attached to a UAV to analyze the blow from small cetaceans and their respiratory microbiome. Preliminary trials to set up the sampling tool were conducted on a group of 6 bottlenose dolphins (Tursiops truncatus) under human care, housed at Acquario di Genova, with approximately 1 meter distance between the blowing animal and the tool to obtain suitable samples. The same sampling kit, suspended via a 2 meter rope assembled on a waterproof UAV, flying 3 meters above the animals, was used to sample the blows of 5 wild bottlenose dolphins in the Gulf of Ambracia (Greece) and a sperm whale (Physeter macrocephalus) in the southern Tyrrhenian Sea (Italy), to investigate whether this experimental assembly also works for large whale sampling. In order to distinguish between blow-associated microbes and seawater microbes, we pooled 5 seawater samples from the same area where blow samples' collection were carried out. The the respiratory microbiota was assessed by using the V3-V4 region of the 16S rRNA gene via Illumina Amplicon Sequencing. The pooled water samples contained more bacterial taxa than the blow samples of both wild animals and the sequenced dolphin maintained under human care. The composition of the bacterial community differed between the water samples and between the blow samples of wild cetaceans and that under human care, but these differences may have been mediated by different microbial communities between seawater and aquarium water. The sperm whale's respiratory microbiome was more similar to the results obtained from wild bottlenose dolphins. Although the number of samples used in this study was limited and sampling and analyses were impaired by several limitations, the results are rather encouraging, as shown by the evident microbial differences between seawater and blow samples, confirmed also by the meta-analysis carried out comparing our results with those obtained in previous studies. Collecting exhaled air from small cetaceans using drones is a challenging process, both logistically and technically. The success in obtaining samples from small cetacean blow in this study in comparison to previous studies is likely due to the distance the sampling kit is suspended from the drone, which reduced the likelihood that the turbulence of the drone propeller interfered with successfully sampling blow, suggested as a factor leading to poor success in previous studies.

Uptake and translocation of perfluoroalkyl acids (PFAA) in red chicory (Cichorium intybus L.) under various treatments with pre-contaminated soil and irrigation water.

Perfluoroalkyl acids (PFAAs), particularly short-chained ones, have high potential for crop uptake, posing a threat to human health in contaminated areas. There is a scarcity of studies using contaminated water as the medium for PFAAs delivery to crops, and a lack of data on the partitioning of PFAA mixtures in growing media. In this context, a controlled experimental study was carried out in a greenhouse to investigate the uptake of a PFAA mixture into red chicory, a typical crop from a major PFAA contamination hot-spot in northern Italy, under treatments with environmentally relevant concentrations in spiked irrigation water and soil, separately and simultaneously. To our knowledge, this is the first study involving multiple exposure media and laboratory adsorption/desorption batch tests as a way of assessing the decrease in the bioavailability of PFAAs from soil. Exposure concentrations for each of the 9 utilized PFAAs were 0, 1, 10 and 80 µg/L in irrigation water and 0, 100 and 200 ng/gdw in soil, combined into 12 treatments. The highest bioaccumulation was measured for PFBA in roots (maximum of 43 µg/gdw), followed by leaves and heads of the chicory plants in all treatments, with the concentrations exponentially decreasing with an increasing PFAA chain length in all plant compartments. The use of irrigation water as the delivery medium increased the transport of PFAAs to the aerial chicory parts, long-chain substances in particular. Additionally, the distribution of PFAAs in the soil was assessed by depth and compared with laboratory measured soil-water equilibrium partition coefficients, revealing only partial dependency of PFAAs bioavailability on the adsorption in soil.

Reactions of diazo compounds with alkenes catalysed by [RuCl(cod)(Cp)]: effect of the substituents in the formation of cyclopropanation or metathesis products.

The reaction of diazo compounds with alkenes catalysed by complex [RuCl(cod)(Cp)] (cod = 1,5-cyclooctadiene, Cp = cyclopentadienyl) has been studied. The catalytic cycle involves in the first step the decomposition of the diazo derivative to afford the reactive [RuCl(Cp){=C(R(1))R(2)}] intermediate and a mechanism is proposed for this step based on a kinetic study of the simple coupling reaction of ethyl diazoacetate. The evolution of the Ru-carbene intermediate in the presence of alkenes depends on the nature of the substituents at both the diazo N(2)=C(R(1))R(2) (R(1), R(2) = Ph, H; Ph, CO(2)Me; Ph, Ph; C(R(1))R(2) = fluorene) and the olefin substrates R(3)(H)C=C(H)R(4) (R(3), R(4) = CO(2)Et, CO(2)Et; Ph, Ph; Ph, Me; Ph, H; Me, Br; Me, CN; Ph, CN; H, CN; CN, CN). A remarkable reactivity of the complex was recorded, especially towards unstable aryldiazo compounds and electron-poor olefins. The results obtained indicate that either cyclopropanation or metathesis products can be formed: the first products are favoured by the presence of a cyano substituent at the double bond and the second ones by a phenyl.

How a tertiary diamine molecule chelates the silicon dimers of the Si(001) surface: a real-time scanning tunneling microscopy study.

The patterning of silicon surfaces by organic molecules emerges as an original way to fabricate innovative nanoelectronic devices. In this regard, we have studied how a diamine, N,N,N',N'-tetramethylethylenediamine (TMEDA, (CH3)2N-[CH2]2-N(CH3)2), chelates the silicon dimers of the Si(001)-2 × 1 surface. Starting from very low coverage to surface saturation (at 300 K), we used real-time scanning tunneling microscopy (STM) in a scanning-while-dosing approach. The images show that the molecules can adopt two bonding configurations: the cross-trench (CT) configuration by bridging two adjacent dimer rows, and the end-bridge (EB) configuration by chelating two adjacent dimers in the same row. However, while CT dominates over EB at low coverage, the percentage of EB adducts steadily increases, until it becomes largely dominant at high molecular coverage. Above a critical coverage θmol of ∼0.13 monolayer (ML), a sudden change in the molecular imprints is seen, likely due to a change in the tunneling conditions. The EB stapling of two adjacent dimers in a row via a dual-dative bond (as shown by XPS) is achieved efficiently by the TMEDA molecule with a very high chemical selectivity. The EB is a unique configuration in amine adsorption chemistry, as it leads to the formation of a pair of first-neighbor, doubly-occupied dangling bonds. Further reactivity of the EB site with other molecules remains to be explored, and possible reaction schemes are envisaged.