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A biomonitoring approach based on a single model species cannot be representative of the contaminations impacts on the ecosystem overall. As part of the Interreg DIADeM program (“Development of an integrated approach for the diagnosis of the water quality of the River Meuse”), a study was conducted to establish the proof of concept that the use of a multispecies active biomonitoring approach improves diagnostic of aquatic systems. The complementarity of the biomarker responses was tested in four model species belonging to various ecological compartments: the bryophyte Fontinalis antipyretica, the bivalve Dreissena polymorpha, the amphipod Gammarus fossarum and the fish Gasterosteus aculeatus. The species have been caged upstream and downstream from five wastewater treatment plants (WWTPs) in the Meuse watershed. After the exposure, a battery of biomarkers was measured and results were compiled in an Integrated Biomarker Response (IBR) for each species. A multispecies IBR value was then proposed to assess the quality of the receiving environment upstream the WWTPs. The effluent toxicity was variable according to the caged species and the WWTP. However, the calculated IBR were high for all species and upstream sites, suggesting that the water quality was already downgraded upstream the WWTP. This contamination of the receiving environment was confirmed by the multispecies IBR which has allowed to rank the rivers from the less to the most contaminated. This study has demonstrated the interest of the IBR in the assessment of biological impacts of a point-source contamination (WWTP effluent) but also of the receiving environment, thanks to the use of independent references. Moreover, this study has highlighted the complementarity between the different species and has emphasized the interest of this multispecies approach to consider the variability of the species exposition pathway and sensibility as well as the mechanism of contaminants toxicity in the final diagnosis. DIADEM

This deliverable is a report to provide information and recommendations on the monitoring strategy of Argo in transitional areas between the coastal shelf seas and the open ocean zone taking into account the collaboration framework of Euro-Argo ERIC with other Marine RIs and research communities.

Fish development and acclimation to environmental conditions are strongly mediated by the hormonal endocrine system. In environments contaminated by anthropogenic stressors, hormonal pathway alterations can be detrimental for growth, survival, fitness, and at a larger scale for population maintenance. In the context of increasingly contaminated marine environments worldwide, numerous laboratory studies have confirmed the effect of one or a combination of pollutants on fish hormonal systems. However, this has not been confirmed in situ. In this review, we explore the body of knowledge related to the influence of anthropogenic stressors disrupting fish endocrine systems, recent advances (focusing on thyroid hormones and stress hormones such as cortisol), and potential research perspectives. Through this review, we highlight how harbours can be used as "in situ laboratories" given the variety of anthropogenic stressors (such as plastic, chemical, sound, light pollution, and invasive species) that can be simultaneously investigated in harbours over long periods of time.

1. Subjects and experimental design Thirty male starlings (Sturnus vulgaris) were wild caught as adults in Normandy (France) in November 2014. All animals were housed in two large indoor aviaries (L x W x H: 2.2 x 1.4 x 2.1 m) at the University of Antwerp with food and water ad libitum with artificial light dark cycle. Starting from January 2013, all birds were kept in a long day photoperiod (16L/8D) in order to remain photorefractory. The housing and experimental procedures were performed in agreement with the Belgian and Flemish laws and were approved by the Committee on Animal Care and Use of the University of Antwerp, Belgium (2014-52). Starlings were divided into two groups: a hypothyroid group (N=16) and a control group (N=14). The study was started when all birds were photorefractory. Then they were switched from long to short (8L:16D) days to induce the return to photosensitivity. Methimazole (MMI) treatment was started in one group to induce hypothyroidism. By supplementing the drinking water with 0.05% MMI, the endogenous stock of THs gradually decreased until it was fully depleted after 2-3 weeks. MMI treatment was continued for the remainder of the experiment. After 12 weeks of short days, the photosensitive starlings were switched back to long days (16L:8D) so that they became photostimulated. In parallel the control group was exposed to the same photoperiodic regime without receiving any hormone manipulation. We monitored the neuroplasticity repeatedly at 6 different time points. The first time point was at the end of the photorefractory state (PR). After switching to short days we measured every 4 weeks to follow up the song control system plasticity during the photosensitive period (SD4, SD8, SD12). Additionally, we measured after 1 week of long days, as it is known that exposure to 1 long day can already affect TH. Finally, we measured after 4 weeks on long days (LD4), when starlings were fully photostimulated. At each time point, songs were recorded, blood samples were taken, and MRI (DTI and 3D) was acquired. In addition, body weight and beak color were registered. 2. MRI data acquisition The birds were initially anesthetized using 2% Isoflurane (Isoflo ®, Abbot Laboratories Ltd.) in a mixture of 30% O2 and 70% N2 at a flow rate of 600 ml/min. Throughout the entire imaging procedure, respiration rate was monitored with a small pneumatic sensor (SA Instruments, NY, USA) positioned under the bird. Depending on the breathing rate, the anesthetic dose was lowered, ranging between 1% - 2% isoflurane. Body temperature was monitored with a cloacal temperature probe and kept within narrow physiological ranges (41.0 ± 0.2 °C) using a warm air system with a feedback unit (SA Instruments, NY, USA). All MRI measurements were performed on a 7T horizontal MR system (Pharmascan 70/16 US, Bruker Biospin, Germany). Each imaging session started with a T2-weighted 3D anatomical RARE scan (TR: 2000 ms; TE: 11 ms; RARE factor: 8; zero-filled to a matrix of (256x92x64) with voxel resolution (0.089x0.25x0.25) mm³). Subsequently, a 4 shot SE-EPI DTI scan (TR: 7000 ms; TE: 23 ms; d 4ms, D 12ms; b-value 670 s/mm²; 60 diffusion gradient directions; spatial resolution: (0.179x0.179x0.35) mm³; 28 coronal slices) was acquired. After the imaging procedure, birds were left to recover in a warmed recovery box before returning to the aviary. 3. MRI data processing Diffusion data were analyzed with MRtrix3 version 3.0 (Tournier et al., 2012) following the same processing steps as described in (Orije et al., 2021b). Preprocessing of the individual DW-images included following steps: denoising (Veraart et al., 2016), correction for Gibbs ringing (Kellner et al., 2016), motion and distortion correction using FSL (Jenkinson et al., 2012), bias field correction using ANTS (Advanced Normalization Tool; Avants et al. (2010)), whole brain extraction and upsampling to isotropic voxels of 1.75 mm. These preprocessed diffusion weighted images were used to calculate individual diffusion maps (fractional anisotropy (FA), mean, axial and radial diffusivity) and fiber orientation distribution (FOD) images. The neuroanatomical contrast of the individual FA maps allowed the delineation of different ROI’s (Area X and RA) to determine their volume changes using AMIRA software (De Groof et al., 2006). The calculation of FOD images requires global intensity normalization, fiber response function estimation using the unsupervised Dhollander algorithm (Dhollander et al., 2019) and spherical deconvolution (Jeurissen et al., 2014). These FOD images were normalized to create an unbiased study-based FOD template, which involves linear and non-linear registration (Raffelt et al., 2011). Fiber density (FD) and fiber-bundle cross-section (FC) were estimated from the normalized FOD images. The transformation parameters derived from building the FOD template were also applied to the diffusion maps to warp them into the template space to perform voxel-based analysis. Next, these images were smoothed to double voxel size (3.5 x 3.5 x 3.5 mm³). Finally, all normalized diffusion maps were averaged to create a FA template that is used as background to display the statistical results. Thyroid hormones clearly play a role in the seasonal regulation of reproduction, but any role they might play in song behavior and the associated seasonal neuroplasticity in songbirds remains to be elucidated. To pursue this question, we first established seasonal patterns in the expression of thyroid hormone regulating genes in male European starlings employing in situ hybridization methods. Thyroid hormone transporter LAT1 expression in the song nucleus HVC was elevated during the photosensitive phase, pointing towards an active role of thyroid hormones during this window of possible neuroplasticity. In contrast, DIO3 expression was high in HVC during the photostimulated phase, limiting the possible effect of thyroid hormones to maintain song stability during the breeding season. Next, we studied the effect of hypothyroidism on song behavior and neuroplasticity using in vivo MRI. Both under natural conditions as with methimazole treatment, circulating thyroid hormone levels decreased during the photosensitive period, which coincided with the onset of neuroplasticity. This inverse relationship between thyroid hormones and neuroplasticity was further demonstrated by the negative correlation between plasma T3 and the microstructural changes in several song control nuclei and cerebellum. Furthermore, maintaining hypothyroidism during the photostimulated period inhibited the increase in testosterone, confirming the role of thyroid hormones in activating the hypothalamic–pituitary–gonadal (HPG) axis. The lack of high testosterone levels influenced the song behavior of hypothyroid starlings, while the lack of high plasma T4 during photostimulation affected the myelination of several tracts. Potentially, a global reduction of circulating thyroid hormones during the photosensitive period is necessary to lift the brake on neuroplasticity imposed by the photorefractory period, whereas local fine-tuning of thyroid hormone concentrations through LAT1 could activate underlying neuroplasticity mechanisms. Whereas an increase in circulating T4 during the photostimulated period potentially influences the myelination of several white matter tracts, which stabilizes the neuroplastic changes. Given the complexity of thyroid hormone effects, this study is a steppingstone to disentangle the influence of thyroid hormones on seasonal neuroplasticity. Subject 5 and subject 16 died after the second and fourth time point respectively, so only MRI data later time points could not be acquired. Scans of subject 7 at time point 4 failed due to excessive movement.

Dans la Méthode thérapeutique à Glaucon, Galien consacre un chapitre aux "Causes et traitement du cancer et de l'léphantiasis". Concernant la diète, il écrit: "Parmi les poissons, donnez ceux de roche". Le présent article s'intéresse à cette prescription dans les consultations ayant trait au 'cancer" qui furent publiées à Padoue à l'aube des temps modernes. Le terme "cancer" et la localisation des organes affectés (sein, utérus, foie, narine, lèvre) sont expliqués en référence aux connaissances patho-physiologiques de l'époque. L'observation de Galien au sujet du "poisson de roche" est spécifiée dans son traité Sur les facultés des aliments et l'exclusion d'autres types de poissons est justifiée par leur nature "visqueuse". La seconde partie de l'article traite de cette question: le régime padouan pour le traitement du cancer était-il similaire à celui prescrit pour d'autres maladies? Parmi ces dernières sont mentionnées: l'arthrite, des perturbations liées aux organes reproducteurs, "les pierres aux reins ou à la vésicule", "le mal de tête avec tintement", etc. Le brochet, la daurade, le rouget sont au contraire considérés comme une alimentation sanitaire. In the Therapeutics, to Glaucon, Galen devotes a chapter to "The causes and treatment of cancer and elephantiasis". As for diet, he writes: "Give rockfish". This article deals with the dietary recommendations concerning "rockfish" in the consilia relating to "cancer" and which were published in Padua in early modern times. The term "cancer" and the localization of the organs which are affected (breast, uterus, liver, nostril, lip) are explained with reference to the patho-physiological knowledge of the time. Galen's statement about "rockfish" is specified in his On the Powers of Foods, and the exclusion of other fish is justified by their "viscous" nature. The second part of the article is devoted to this question: was the Paduan dietary regime for the treatment of cancer similar to that prescribed for other diseases? Among the latter are metnioend: arthritis, fisturbances linked with the reproductive organs, "stones in the kidneys and in the gall-bladder", "headache with tinnitus", etc. Pike, sea bream, red mullet are on the contrary considered to be healthy food.

This dataset contains data on future changes in sea-surface water properties along the global distribution of mangrove forests. This includes the coordinates of mangrove occurence and associated information on present (2000-2014) and future (2090-2100) sea-surface temperature (SST), sea-surface salinity (SSS), and sea-surface density (SSD, derived from SST and SSS, using the UNESCO EOS-80 equation of state polynomial for seawater - 'sw_dens.m' in MATLAB). SST and SSS fields from which the data were extracted, are from the Bio-ORACLE database. Additionally, for each of the mangrove occurence points (longitude, latitude), information is provided about the main mangrove biogeographical region and associated province as provided in the Marine Ecoregions of the World dataset (Spalding et al., 2007, BioScience), alowing for a spatial analysis of the data. More information and details about the data are provided in the README sheet of the Excel data file. For details about the data, we refer to the README sheet that is provided in the Excel data file, as well as the Methods section in the associated manuscript.

Nursing Students’ Attitudes Regarding Euthanasia Due to Unbearable Mental Suffering: A cross-sectional Study Abstract Background: There is an increasing debate on euthanasia worldwide (e.g. Portugal), with some countries, such as Belgium, altering the legal framework. The euthanasia legislation in certain countries notes that 'unbearable suffering' of a mental nature may also be considered grounds for euthanasia. If such legislation becomes standard practice in the field, the consequences for healthcare workers would be significant. Since nursing students are the nurses of the future, it is important to examine their attitudes towards euthanasia due to unbearable mental suffering (UMS-euthanasia). Objective: The current study introduces the adaptation of the validated Euthanasia Attitude Scale (EAS) with a focus on UMS-euthanasia in the Flemish (Belgium) setting, as well as final year nursing students’ attitudes towards UMS-euthanasia. Design: Cross-sectional survey. Setting/ participants: A sample of final-year baccalaureate nursing students (n=273) from eight of the eleven Flemish university colleges that agreed to participate. Methods: Explorative, descriptive cross-sectional study conducted using an e-mail survey. The actual questionnaire contains 21 questions and was developed based on a consensus reached following independent translations by a native English speaker, a health education expert, and an end-of-life specialist. The psychometric properties of the EAS were assessed, including reliability and validity. Independent-Samples T Test was used to investigate relation between demographic and education-related data, and domain and total score of the UMS-EAS-NL. This study received ethical approval from the Ethical Committee of the University Hospital Brussels. This study was not registered. Results: McDonald's Omega was 0.838 for the total EAS scores, supporting the validity of the questionnaire. There is a statistically significant difference in total questionnaire score between those who have been involved in euthanasia and those who have not (95% CI 0.031–0.511), as well as for ‘ethical considerations’ (95% CI 0.064–0.545). There are clinically important results for ‘Treasuring Life’ and ‘Naturalistic beliefs’. Conclusions: This study’s sample is representative of the concerned population. McDonald's Omega quantifies the reliability allowing for some sub-domains. However, in order to compare with similar studies, the Cronbach's alpha was also calculated, and it is similar to previous studies (α=.812). Most of the final-year nursing students supported the availability of UMS-euthanasia. In order to monitor adequate care, it is necessary to prepare nursing students adequately for this complex matter.

The extraction of volatiles from perishable food at a sub-ambient temperature using headspace solid-phase microextraction (HS-SPME) has not been considered in the past due to the corresponding loss in sensitivity. We propose HS-SPME sampling under vacuum (Vac-HS-SPME) to compensate problems of sensitivity loss and achieve substantial improvement in extraction efficiencies whilst sampling at temperatures as low as 5 °C. The approach was applied to fish samples, representing a highly vulnerable perishable food sample. The theoretical considerations explaining the performance of Vac-HS-SPME at sub-ambient temperatures are discussed and related to the increase in gas diffusivities when sampling under vacuum. A comparative study between Vac- and regular HS-SPME for the extraction of 18 compounds from salmon was carried out at different temperatures (5, 30 and 40 °C) and sampling times (10-60 min). For the majority of the compounds, Vac-HS-SPME at 5 °C yielded similar or superior extraction efficiencies than regular HS-SPME even when sampling at 40 °C. However, four compounds were better extracted at 1 atm presumably due to the intensification of competitive adsorption of analytes on the SPME fiber under vacuum or the partial losses of more volatile analytes during air-evacuation in the presence of the frozen samples. Sub-ambient temperature sampling (5 °C) combined with Vac-HS-SPME was also applied to monitor the changes in the 18 compounds present in salmon, redfish, and cod refrigerated for up to five days. The results were compared to those obtained with regular HS-SPME at 40 °C. Overall, Vac-HS-SPME sampling at 5 °C represents a new and powerful approach for the analysis of volatiles in refrigerated foods, and has a great potential for future studies in quality control and freshness assessment.

Spatio‐temporal variability of marine soundscapes reflects environmental dynamics and local habitat health. This study characterizes the coastal soundscape of the Cres‐Lošinj Natura 2000 Site of Community Importance, encompassing the non‐tourist (11–15 March 2020) and the tourist (26–30 July 2020) season. A total of 240 h of continuous recordings were manually analyzed and the abundance of animal vocalizations and boat noise was obtained; sound pressure levels were calculated for the low (63–2000 Hz) and high (2000–20,000 Hz) frequency range. Two fish sound types were drivers of both seasonal and diel variability of the low‐frequency soundscape. The first is emitted by the cryptic Roche’s snake blenny (Ophidion rochei), while the second, whose emitter remains unknown, was previously only described in canyons and coralligenous habitats of the Western Mediterranean Sea. The high‐frequency bands were characterized by bottlenose dolphin (Tursiops truncatus) vocalizations, indicating dolphins’ use of area for various purposes. Boat noise, however, dominated the local soundscape along the whole considered periods and higher sound pressure levels were found during the Tourist season. Human‐generated noise pollution, which has been previously found 10 years ago, is still present in the area and this urges management actions. Interreg Italy-Croatia CBC Programme