Regional evaluation of glyphosate pollution in the minor irrigation network.

Due to its low cost, its ease of use and to the "mild action" declared for long time by the Control and Approval Agencies towards it, the herbicide Glyphosate, is one of the currently best-selling and most-used agricultural products worldwide. In this work, we evaluated the presence and spread of Glyphosate in the Po River Basin (Northern Italy), one of the regions with the most intensified agriculture in Europe and where, by now for decades, a strong and general loss of aquatic biodiversity is observed. In order to carry out a more precise study of the real presence of this herbicide in the waters, samples were collected from the minor water network for two consecutive years, starting in 2022, at an interval time coinciding with those of the spring and summer crop treatments. In contrast to the sampling strategies generally adopted by Environmental Protection Agencies, a more focused sampling strategy was adopted to highlight the possible high concentrations in minor watercourses in direct contact with cultivated fields. Finally, we investigated the possible consequences that the higher amounts of Glyphosate found in our monitoring activities can have on stress reactions in plant (Groenlandia densa) and animal (Daphnia magna) In all the monitoring campaigns we detected exceeding European Environmental Quality Standard - EQS limits (0.1 µg/L) values. Furthermore, in some intensively agricultural areas, concentrations reached hundreds of µg/L, with the highest peaks during spring. In G. densa and D. magna, the exposition to increasing doses of herbicide showed a clear response linked to metabolic stress. Overall, our results highlight how, after several decades of its use, the Glyphosate use efficiency is still too low, leading to economic losses for the farm and to strong impacts on ecosystem health. Current EU policy indications call for an agroecological approach necessary to find alternatives to chemical weed control, which farms can develop in different contexts in order to achieve the sustainability goals set by the Farm to Fork strategy.

Data for the stress update assessment in large-deformation finite element analysis.

During a research work about stress integration schemes for large-deformation finite element analysis many datasets are collected from both numerical and analytical models. The corresponding numerical data (stress and displacements) are computed by means of different finite element formulations including the well-established stress update schemes employed by the major commercial software packages. To this purpose a suitable finite element code, capable of easily switching the different methods, is implemented. Accordingly, the data computed for three stress integration tests allowing analytical solution in the case of linear material are presented. The comparison of all the predictions from the various methods allows the choice of the most accurate model in predicting displacement and related stress. In addition, the data may be reused as starting point in the development of new stress integration strategies, as a reference comparison to understand the behaviour of the standard methods.

Limited contribution of hatchery-produced individuals to the sustainment of wild marble trout (Salmo marmoratus Cuvier, 1829) in an Alpine basin.

Fish stocking constitutes a common management practice in freshwaters all over the world, to enhance fisheries or to support threatened fish populations. Pervasive detrimental effects may affect the real effectiveness of stocking programs. However, studies assessing the real impacts and relative contribution of stocked trout in wild populations are surprisingly few. The marble trout, Salmo marmoratus (Cuvier 1829), is a critically endangered sub-endemic salmonid in Northern Italy, and an iconic species for recreational fishing and conservation, also representing an emblematic case of species negatively affected by restocking. For instance, marble trout inhabiting the Toce River, the second largest tributary of Lake Maggiore, has been stocked with different hatchery congener trout belonging to the Salmo trutta complex (putative marble trout, Atlantic trout Salmo trutta Linnaeus 1758 and putative Mediterranean trout Salmo ghigii Pomini 1941) over the last decades. Using mitochondrial (D-loop) and nuclear (12 microsatellites and LDH-C1*) markers, we characterised the genetic variability and gene flow among the wild and hatchery individuals of marble trout of this basin, to investigate the effectiveness of stocking activities on the native residual population. Despite extensive hybridization of marble trout with non-native brown trout stocks was shown, the presence of individuals belonging to pure native stock has been detected as well. However, concerns could be advanced regarding its future persistence, due to climatic and hydraulic instabilities or loss of environmental heterogeneity. Moreover, despite ongoing yearly massive stocking activities, a negligible contribution of reared putative marble trout in the wild sample has been documented, suggesting that natural recruitment represents the greatest source of this wild population sustainment. Important adaptive differences between wild and domestic trout are present, likely due to the deleterious long-term effects of the close breeding system of hatcheries. Finally, possible implications for stocking management improvement have been discussed.

Suspect screening of wastewaters to trace anti-COVID-19 drugs: Potential adverse effects on aquatic environment.

During the first period of the SARS-CoV-2 pandemic, the lack of specific therapeutic treatments led to the provisional use of a number of drugs, with a continuous review of health protocols when new scientific evidence emerged. The management of this emergency sanitary situation could not take care of the possible indirect adverse effects on the environment, such as the release of a large amount of pharmaceuticals from wastewater treatment plants. The massive use of drugs, which were never used so widely until then, implied new risks for the aquatic environment. In this study, a suspect screening approach using Liquid Chromatography-High Resolution Mass Spectrometry techniques, allowed us to survey the presence of pharmaceuticals used for COVID-19 treatment in three WWTPs of Lombardy region, where the first European cluster of SARS-CoV-2 cases was detected. Starting from a list of sixty-three suspect compounds used against COVID-19 (including some metabolites and transformation products), six compounds were fully identified and monitored together with other target analytes, mainly pharmaceuticals of common use. A monthly monitoring campaign was conducted in a WWTP from April to December 2020 and the temporal trends of some anti-COVID-19 drugs were positively correlated with those of COVID-19 cases and deaths. The comparison of the average emission loads among the three WWTPs evidenced that the highest loads of hydroxychloroquine, azithromycin and ciprofloxacin were measured in the WWTP which received the sewages from a hospital specializing in the treatment of COVID-19 patients. The monitoring of the receiving water bodies evidenced the presence of eight compounds of high ecological concern, whose risk was assessed in terms of toxicity and the possibility of inducing antibiotic and viral resistance. The results clearly showed that the enhanced, but not completely justified, use of ciprofloxacin and azithromycin represented a risk for antibiotic resistance in the aquatic ecosystems.

Functional response of fish communities in a multistressed freshwater world.

Freshwater fish communities are impacted by multiple pressures, determining loss of functional diversity and redundancy. Our aim was to disentangle the roles and relevancies of different pressures in shaping fish communities in small streams of the Po plain (North Italy). Long term trend (1998-2018) of functional diversity of 31 fish communities was assessed and modeled in respect to three potential pressures: temperature increase, intensity of exotic fish invasion, and habitat quality degradation. Ecological traits mostly influenced by the pressures were also identified. Reduction of functional richness mostly due to local extinction or contraction of cold adapted predators, such as salmonids, was linked to increasing temperatures. Warming probably also led to a shift of generalist and dominant species, which became more abundant in streams hosting mixed communities of salmonids and cyprinids, and determined the increase of functional dispersion and uniqueness. Reduction of functional redundancy and increasing functional dispersion were both also related to the introduction of new ecological traits brought by expanding exotic species. Low functional overlap was found among native and exotic species, indicating that the invasion process was mainly controlled by competitive interactions and/or resource opportunism. Functional response to habitat quality was not clearly evident. In conclusion, the impact of temperature increase and exotic species on fish functional diversity was effective, idiosyncratic and mediated by the scale of analysis and by the intensity of pressures.

Presence and infectivity of SARS-CoV-2 virus in wastewaters and rivers.

The presence of SARS-CoV-2 in raw wastewaters has been demonstrated in many countries affected by this pandemic. Nevertheless, virus presence and infectivity in treated wastewaters, but also in the receiving water bodies are still poorly investigated. In this study, raw and treated samples from three wastewater treatment plants, and three river samples within the Milano Metropolitan Area, Italy, were surveyed for SARS-CoV-2 RNA detection by means of real time RT-PCR and infectivity test on culture cells. SARS-CoV-2 RNA was detected in raw, but not in treated wastewaters (four and two samples, respectively, sampled in two dates). The isolated virus genome was sequenced, and belonged to the strain most spread in Europe and similar to another found in the same region. RNA presence in raw wastewater samples decreased after eight days, probably following the epidemiological trend estimated for the area. Virus infectivity was always null, indicating the natural decay of viral pathogenicity in time from emission. Samples from receiving rivers (three sites, sampled in the same dates as wastewaters) showed in some cases a positivity to real time RT-PCR, probably due to non-treated, or inefficiently treated discharges, or to the combined sewage overflows. Nevertheless, also for rivers infectivity was null. Risks for public health should be limited, although a precautionary approach to risk assessment is here advocated, giving the preliminary nature of the presented data.

Bovine lymph nodes as a source of Escherichia coli contamination of the meat.

Ground beef contamination with Escherichia coli is usually a result of carcass faecal contamination during the slaughter process. Carcasses are contaminated when they come into contact with soiled hides or intestinal leakage content during dressing and the evisceration processes. A more recent and compelling hypothesis is that, when lymph nodes are present in manufacturing beef trimmings, they can be a potential source of Enterobacteriaceae contamination of ground beef. The aim of this study was to investigate the occurrence of E. coli in lymph nodes from beef carcasses used for ground meat production, in six slaughter plants situated in central Italy A total of 597 subiliac (precrural) lymph nodes were obtained from 597 cattle carcasses and screened for E. coli by culture. Furthermore, E. coli isolates (one per positive carcass) were tested for stx1, stx2 eaeA and hlyA genes that are commonly used to identify and characterise shiga toxin-producing E. coli (STEC). In addition, the E. coli isolates were profiled for antimicrobial susceptibility. A proportion of 34.2% (204/597) carcasses were positive for E. coli. PCR revealed that 29% (59/204) of E. coli possessed stx1 or stx2 which corresponded to 9.9% of the cattle sampled. Moreover, a combination of stx1 or stx2 and eaeA was found in in 4 isolates (2% among E. coli positive samples and 1% among cattle sampled) and a combination of stx1 or stx2 and eaeA and hly in 1 isolate (0.5% and 0.2%). More than 95% of isolates were susceptible to gentamicin, ceftriaxone, cyprofloxacin and cefotaxime while high rates of resistance were recorded for cephalotin, ampicillin, tetracycline, tripe sulfa and streptomycin. The multivariate analysis identified "age" as the factor most closely related to E. coli positivity (either generic E. coli or STEC) in bovine lymph nodes. In conclusion, subiliac lymph nodes represent a source of E. coli for ground beef. These results are of major importance for risk assessment and improving good manufacturing practices during animal slaughter and ground meat production.

Water and microbial monitoring technologies towards the near future space exploration.

Space exploration is demanding longer lasting human missions and water resupply from Earth will become increasingly unrealistic. In a near future, the spacecraft water monitoring systems will require technological advances to promptly identify and counteract contingent events of waterborne microbial contamination, posing health risks to astronauts with lowered immune responsiveness. The search for bio-analytical approaches, alternative to those applied on Earth by cultivation-dependent methods, is pushed by the compelling need to limit waste disposal and avoid microbial regrowth from analytical carryovers. Prospective technologies will be selected only if first validated in a flight-like environment, by following basic principles, advantages, and limitations beyond their current applications on Earth. Starting from the water monitoring activities applied on the International Space Station, we provide a critical overview of the nucleic acid amplification-based approaches (i.e., loop-mediated isothermal amplification, quantitative PCR, and high-throughput sequencing) and early-warning methods for total microbial load assessments (i.e., ATP-metry, flow cytometry), already used at a high readiness level aboard crewed space vehicles. Our findings suggest that the forthcoming space applications of mature technologies will be necessarily bounded by a compromise between analytical performances (e.g., speed to results, identification depth, reproducibility, multiparametricity) and detrimental technical requirements (e.g., reagent usage, waste production, operator skills, crew time). As space exploration progresses toward extended missions to Moon and Mars, miniaturized systems that also minimize crew involvement in their end-to-end operation are likely applicable on the long-term and suitable for the in-flight water and microbiological research.

Embryo/larval toxicity and transcriptional effects in zebrafish (Danio rerio) exposed to endocrine active riverbed sediments.

Sediment toxicity plays a fundamental role in the health of inland fish communities; however, the assessment of the hazard potential of contaminated sediments is not a common objective in environmental diagnostics or remediation. This study examined the potential of transcriptional endpoints investigated in zebrafish (Danio rerio) exposed to riverbed sediments in ecotoxicity testing. Embryo-larval 10-day tests were conducted on sediment samples collected from five sites (one upstream and four downstream of the city of Milan) along a polluted tributary of the Po River, the Lambro River. Sediment chemistry showed a progressive downstream deterioration in river quality, so that the final sampling site showed up to eight times higher concentrations of, for example, triclosan, galaxolide, PAH, PCB, BPA, Ni, and Pb, compared with the uppermost site. The embryo/larval tests showed widespread toxicity although the middle river sections evidenced worse effects, as evidenced by delayed embryo development, hatching rate, larval survival, and growth. At the mRNA transcript level, the genes encoding biotransformation enzymes (cyp1a, gst, ugt) showed increasing upregulations after exposure to sediment from further downstream sites. The genes involved in antioxidant responses (sod, gpx) suggested that more critical conditions may be present at downstream sites, but even upstream of Milan there seemed to be some level of oxidative stress. Indirect evidences of potential apoptotic activity (bcl2/bax < 1) in turn suggested the possibility of genotoxic effects. The genes encoding for estrogen receptors (erα, erß1, erß2) showed exposure to (xeno)estrogens with a progressive increase after exposure to sediments from downstream sites, paralleled by a corresponding downregulation of the ar gene, likely related to antiandrogenic compounds. Multiple levels of thyroid disruption were also evident particularly in downstream zebrafish, as for thyroid growth (nkx2.1), hormone synthesis and transport (tg, ttr, d2), and signal transduction (trα, trß). The inhibition of the igf2 gene reasonably reflected larval growth inhibitions. Although none of the sediment chemicals could singly explain fish responses, principal component analysis suggested a good correlation between gene transcripts and the overall trend of contamination. Thus, the combined impacts from known and unknown covarying chemicals were proposed as the most probable explanation of fish responses. In summary, transcriptional endpoints applied to zebrafish embryo/larval test can provide sensitive, comprehensive, and timeliness information which may greatly enable the assessment of the hazard potential of sediments to fish, complementing morphological endpoints and being potentially predictive of longer studies.

Effects of Perfluoralkyl Substances on a Multigenerational Scale: A Case Study with Chironomus riparius (Diptera, Chironomidae).

A multigenerational test with Chironomus riparius was performed to assess long-term effects on life-traits of exposure to selected perfluoroalkyl compounds: perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), and perfluorobutane sulfonate (PFBS). These persistent contaminants are widespread in aquatic ecosystems at low concentrations, possibly exerting long-term toxicity. Larvae of C. riparius of a native population were exposed for 10 generations to 10 µg/L nominal concentrations of PFOS, PFOA, and PFBS, comparable with the maximum values found in European rivers. All treatments showed reduced growth at most/several generations. No effects on survival, development, and reproduction were found. A final tolerance-induction test was performed exposing the pre-exposed experimental cohorts to 100 µg/L PFOS and 150 µg/L PFOA for a whole life cycle. Factorial analysis of variance revealed no difference between treatments (i.e., PFOS vs PFOA), indicating no induced tolerance. Instead, organisms pre-exposed to PFBS were the most stressed, followed by those pre-exposed to PFOA and PFOS, with earlier emergence and reduced adult weight. The results may be related to general stress and genetic erosion induced by long-term laboratory culture, but also to long-term toxicant exposure. However, no effects at the population level (population growth rate) were proved, and thus a toxicity risk in real ecosystems at the tested concentrations seems unlikely. Environ Toxicol Chem 2019;00:1-12. © 2019 SETAC.

Gene expression and genotoxicity in Manila clam (Ruditapes philippinarum) modulated by sediment contamination and lagoon dynamics in the Po river delta.

The lagoons of the Po River delta are potentially exposed to complex mixtures of contaminants, nevertheless, there is a substantial lack of information about the biological effects of these contaminants in the Po delta lagoons. These environments are highly dynamic and the interactions between chemical and environmental stressors could prevent the proper identification of biological effects and their causes. In this study, we aimed to disentangle such interactions focusing on Manila clams, previously exposed to six lagoons of the Po delta, adopting three complementary tools: a) the detailed description via modelling techniques of lagoon dynamics for salinity and water temperature; b) the response sensitivity of a number of target genes (ahr, cyp4, ρ-gst, σ-gst, hsp22, hsp70, hsp90, ikb, dbh, ach, cat, Mn-sod, Cu/Zn-sod, cyp-a, flp, grx, TrxP) investigated in clam digestive glands by Real Time PCR; and c) the relevance of DNA adducts determined in clams as markers of exposure to genotoxic chemicals. The lagoons showed specific dynamics, and two of them (Marinetta and Canarin) could induce osmotic stress. A group of genes (ahr, cyp4, Mn-sod, σ-gst, hsp-22, cyp-a, TrxP) seemed to be associated with overall lagoon characteristics as may be described by salinity and its variations. Lagoon modelling and a second group of genes (hsp70, hsp90, cat, ikb, ach, grx, Cu/Zn-sod) also suggested that moderate increases of river discharge may imply worse exposure conditions. Oxidative stress seemed to be associated with such events but it was slightly evident also under normal exposure conditions. DNA adduct formation was mainly associated with overwhelmed antioxidant defences (e.g. low Cu/Zn-sod) or seemingly with their lack of response in due time. In Po delta lagoons, Manila clam can be affected by chemical and environmental factors which can contribute to induce oxidative stress, DNA adduct formation and, ultimately, to affect clam condition and health.

Hepatic gene expression profiles of a non-model cyprinid (Barbus plebejus) chronically exposed to river sediments.

In this study, we characterized the gene expression responses of the Padanian barbel (Barbus plebejus), a native benthivorous cyprinid with a very compromised presence within the fish community of the River Po. Barbel juveniles were exposed in the laboratory to two river sediments reflecting an upstream/downstream gradient of increasing contamination and collected from one of the most anthropized tributaries of the River Po. After 7months of exposure, hepatic transcriptional changes that were diagnostic of sediment exposure were assessed. We investigated a set of 24 genes involved in xenobiotic biotransformation (cyp1a, gstα, ugt), antioxidant defense (gpx, sod, cat, hsp70), trace metal exposure (mt-I, mt-II), DNA repair (xpa, xpc), apoptosis (bax, casp3), growth (igf2), and steroid (erα, erß1, erß2, ar, vtg) and thyroid (dio1, dio2, trα, trß, nis) hormone signaling pathways. In a consistent overall picture, the results showed that long-term sediment exposure mainly increased the levels of mRNAs encoding proteins involved in xenobiotic metabolism, oxidative stress defense, repair of DNA damage and activation of the apoptotic process. Transcript up-regulation of three receptor genes (erß2, ar, trß), likely representing compensatory responses to antagonistic/toxic effects, was also observed, confirming the exposure to disruptors of the reproductive and thyroidal axes. In contrast to expectations, a few genes showed no response (e.g., casp3) or even downregulation (vtg), further suggesting that the timing of exposure/assessment, potential compensatory effects or post-transcriptional modifications interact to modify the gene expression profiles, particularly during exposure to mixtures of contaminants.

A new sequence data set of SSU rRNA gene for Scleractinia and its phylogenetic and ecological applications.

Scleractinian corals (i.e. hard corals) play a fundamental role in building and maintaining coral reefs, one of the most diverse ecosystems on Earth. Nevertheless, their phylogenies remain largely unresolved and little is known about dispersal and survival of their planktonic larval phase. The small subunit ribosomal RNA (SSU rRNA) is a commonly used gene for DNA barcoding in several metazoans, and small variable regions of SSU rRNA are widely adopted as barcode marker to investigate marine plankton community structure worldwide. Here, we provide a large sequence data set of the complete SSU rRNA gene from 298 specimens, representing all known extant reef coral families and a total of 106 genera. The secondary structure was extremely conserved within the order with few exceptions due to insertions or deletions occurring in the variable regions. Remarkable differences in SSU rRNA length and base composition were detected between and within acroporids (Acropora, Montipora, Isopora and Alveopora) compared to other corals. The V4 and V9 regions seem to be promising barcode loci because variation at commonly used barcode primer binding sites was extremely low, while their levels of divergence allowed families and genera to be distinguished. A time-calibrated phylogeny of Scleractinia is provided, and mutation rate heterogeneity is demonstrated across main lineages. The use of this data set as a valuable reference for investigating aspects of ecology, biology, molecular taxonomy and evolution of scleractinian corals is discussed.

Can Drainage Using a Negative-Pressure Wound Therapy Device Replace Traditional Sample Collection Methods?

BACKGROUND: In 2015 a new device for the collection of mediastinal fluid from patients with deep sternal wound infection (DSWI) in the presence of negative-pressure wound therapy (NPWT) became available. The present study was designed to evaluate whether changing sample collection devices increased micro-organism detection in patients undergoing NPWT. METHODS: During 2013-2014, 207 samples were collected and cultured from NPWT patients (n = 23) to demonstrate the presence of DSWI using reticulated polyurethane sponge culture, a swab, and blood culture. In 2015, a new collection device was introduced for specimen collection. A total of 357 samples (n = 17) were collected using the ESwab(™) (Copan, Murrieta, CA) for deep and superficial wound sample collection. In addition, blood culture devices were used for collecting mediastinal fluid aspirated directly from the wound and biologic fluid obtained from the NPWT device. Fisher exact test was performed to test the rate of independence rate of micro-organism identification using the NPWT sponge device and taking blood culture results as a reference for micro-organism identification. RESULTS: After the introduction of the new collection device in our hospital, an overall increase in the detection of micro-organisms (46.7%) was reported. During 2013-2014 our traditional microbiologic collection method did not detect a pathogen in 30.4% of patients. During 2015, the new sample collection approach, direct from the NPWT device, improved micro-organism detection by 10.4% and reduced DSWIs with undetected pathogens to 17.6% (p < 0.01). CONCLUSIONS: As a result of proficiency gained in the last year, the most representative specimen in wound infection was represented by mediastinal fluid collected directly from the wound and the NPWT device. Given the correlation between the blood culture of micro-organisms detected using the ESwab device from the wound, mediastinal drainage, and drainage from the NPWT device, we can assume that the NPWT device may replace the other biologic sampling devices.

Endocrine-disrupting chemicals in coastal lagoons of the Po River delta: sediment contamination, bioaccumulation and effects on Manila clams.

The large estuary that the River Po forms at its confluence into the Adriatic Sea comprises a multitude of transitional environments, including coastal lagoons. This complex system receives the nutrients transported by the River Po but also its load of chemical contaminants, which may pose a substantial (eco)toxicological risk. Despite the high ecological and economic importance of these vulnerable environments, there is a substantial lack of information on this risk. In light of the recent amendments of the European Water Framework Directive (2013/39/EU), the present study investigated the sediment contamination of six coastal lagoons of the Po delta and its effects on Manila clams (Ruditapes philippinarum), exposed in situ for 3 months. Sediment contamination and clam bioaccumulation of a wide range of chemicals, i.e. trace metals (Cd, Cr, Ni, Hg, Pb, As), polybrominated diphenyl ethers (PBDEs), alkylphenols (APs), organochlorine compounds (PCBs, DDTs), polycyclic aromatic hydrocarbons (PAHs) and organotins (TPhT, TBT), suggested a southward increase related to the riverine transports. Where the River Po influence was more direct, the concentrations of contaminants were higher, with nonylphenol and BDE-209 exceeding sediment quality guidelines. Biometric indicators suggested the influence of contamination on organism health; an inverse relationship between PBDEs in sediments and clam condition index has been found, as well as different biota-sediment accumulation factors (BSAFs) in the lagoons.

Eutrophication management in surface waters using lanthanum modified bentonite: A review.

This paper reviews the scientific knowledge on the use of a lanthanum modified bentonite (LMB) to manage eutrophication in surface water. The LMB has been applied in around 200 environments worldwide and it has undergone extensive testing at laboratory, mesocosm, and whole lake scales. The available data underline a high efficiency for phosphorus binding. This efficiency can be limited by the presence of humic substances and competing oxyanions. Lanthanum concentrations detected during a LMB application are generally below acute toxicological threshold of different organisms, except in low alkalinity waters. To date there are no indications for long-term negative effects on LMB treated ecosystems, but issues related to La accumulation, increase of suspended solids and drastic resources depletion still need to be explored, in particular for sediment dwelling organisms. Application of LMB in saline waters need a careful risk evaluation due to potential lanthanum release.

Evaluating the impact of a fluoropolymer plant on a river macrobenthic community by a combined chemical, ecological and genetic approach.

Effect-based monitoring is a recommended approach suggested in European Guidelines to assess the response of ecosystem affected by a pollution source, considering the effects at community, population, individual but also at suborganism level. A combined chemical, ecological and genetic approach was applied in order to assess the impact of a fluoropolymer plant on the macrobenthic community of the Northern Italian river Bormida (Piedmont region). The macrobenthic community living downstream of the industrial discharge was chronically exposed to a mixture of perfluoroalkyl substances (PFAS), with perfluorooctanoic acid as the main compound, at concentrations up to several µgL(-1). Ecological assessment proved that the downstream community was not substantially different from that living upstream of the pollution source. The impact on community is not quantifiable with the traditional monitoring methods used for ecological classification under European regulation because macrobenthic communities showed only slight differences in their structure. In order to highlight effects on genetic variability of the native population, a subcellular analysis by using the AFLP (Amplified Fragment Length Polymorphism) genetic technique was applied to genotype of individuals of a selected species (Hydropsyche modesta, Trichoptera) collected in the two sampling sites. Percentage of variation between the two populations was 6.8%, a threshold compatible with a genetic drift induced in the downstream population. The genetic study carried out in field identified a significant divergence between exposed and non-exposed populations, but at present it is not possible to associate this divergence to a specific effect induced by PFAS.

Pachyseris inattesa sp. n. (Cnidaria, Anthozoa, Scleractinia): a new reef coral species from the Red Sea and its phylogenetic relationships.

A new scleractinian coral species, Pachyseris inattesa sp. n., is described from the Red Sea. Despite a superficial resemblance with some species in the agariciid genus Leptoseris with which it has been previously confused, P. inattesa sp. n. has micro-morphological characters typical of the genus Pachyseris. This genus, once part of the Agariciidae, is comprised of five extant species and is widely distributed throughout the tropical Indo-Pacific. It is currently incertae sedis as a result of recent molecular analysis and appears to be closely related to the Euphylliidae. A molecular phylogenetic reconstruction including P. inattesa sp. n., the genus type species P. rugosa, and P. speciosa, all present in the Red Sea, was performed using the mitochondrial intergenic spacer between COI and 16S-rRNA. The results confirm that P. inattesa sp. n. is a monophyletic lineage closely related to the other Pachyseris species examined.

DNA barcoding reveals the coral "laboratory-rat", Stylophora pistillata encompasses multiple identities.

Stylophora pistillata is a widely used coral "lab-rat" species with highly variable morphology and a broad biogeographic range (Red Sea to western central Pacific). Here we show, by analysing Cytochorme Oxidase I sequences, from 241 samples across this range, that this taxon in fact comprises four deeply divergent clades corresponding to the Pacific-Western Australia, Chagos-Madagascar-South Africa, Gulf of Aden-Zanzibar-Madagascar, and Red Sea-Persian/Arabian Gulf-Kenya. On the basis of the fossil record of Stylophora, these four clades diverged from one another 51.5-29.6 Mya, i.e., long before the closure of the Tethyan connection between the tropical Indo-West Pacific and Atlantic in the early Miocene (16-24 Mya) and should be recognised as four distinct species. These findings have implications for comparative ecological and/or physiological studies carried out using Stylophora pistillata as a model species, and highlight the fact that phenotypic plasticity, thought to be common in scleractinian corals, can mask significant genetic variation.

Refugia within refugia as a key to disentangle the genetic pattern of a highly variable species: the case of Rana temporaria Linnaeus, 1758 (Anura, Ranidae).

Two distinct lineages of Rana temporaria are known in the Palaearctic region, but it is uncertain whether this species persisted in one or more Pleistocene refugia. We resolved the phylogeographic history and genetic variability of R. temporaria in the Italian peninsula, a 'traditional' Pleistocene refugium, and related our findings to patterns described for other European populations. We sequenced the mitochondrial markers Cox I and cytochrome b. Phylogenetic reconstruction only indicated the presence of haplotypes belonging to the Western lineage in the Italian peninsula. Overall, the genetic variability of Italian populations was higher than other European populations, which shared haplotypes with the Alpine populations. We demonstrated subdivision into five main Italian sublineages, which was associated with a geographical structure of populations in two divergent groups. In particular, one Apennine group might have resulted from bottlenecks during the last interglacials ages. In contrast, Alpine populations were recently diverged and showed incomplete lineage sorting. Our data indicate that the Italian peninsula served as refugium for the Western lineage of R. temporaria. Dispersion towards Central Europe probably started only from the western slope of the Alps via a rapid leading edge expansion. The identified structure is partially congruent with traditional peripheral refugia identified for plants. This evolutionary scenario does not support any taxonomic distinction at the subspecific level for R. temporaria.