Abed El

Rahman Hassoun
email: abedhassoun@cnrs.edu.lb

Joel Guiot

Katarzyna Marini

Wolfgang Cramer

THE CHANGING MEDITERRANEAN BASIN THROUGH THE LENS OF MEDITERRANEAN EXPERTS

Keywords: climate change, environmental change, resources, ecosystems, adaptation, society

The different components of the Mediterranean Basin (land, coast, and sea) are undergoing significant changes due to multiple anthropogenic pressures, including climate change, pollution and other factors. The 1 st Mediterranean Assessment Report (MAR1), produced by the Mediterranean Experts on Climate and environmental Change (MedECC) and based on available scientific literature, provides a coherent and comprehensive synthesis of the status of the Mediterranean Basin, its main drivers and their impacts on both ecosystems and human dimensions, with a focus on water, food, energy, ecosystems and ecosystem services, development, health and human security. The report is the first multidisciplinary, trans-boundary assessment produced for this region and provides significant input to inform policy at regional, national and local levels. Its groundbreaking nature lies in the scope of the scientific assessment as well as in displaying the regional capacity to coordinate existing fragmented efforts in the region. The report highlights risks and possible response strategies that may help to increase the resilience to the effects of climate and environmental change. The conclusions show that effective policy responses, as well as in support of Sustainable Development Goals, encompass both strengthened mitigation of climate and environmental change and enhanced adaptation to their impacts. Socio-economic factors of poverty, inequalities and gender imbalances presently hamper the achievement of sustainable development and climate resilience in Mediterranean countries.

INTRODUCTION

All sub-regions of the Mediterranean Basin, on land and in the sea, are impacted by recent anthropogenic changes in the environment (MedECC 2020). Greenhouse gases emissions, generated by various anthropogenic activities since the industrial revolution, are changing the climate patterns worldwide, with exacerbated trends in the Mediterranean compared to other areas [START_REF] Cramer | Climate change and interconnected risks to sustainable development in the Mediterranean[END_REF]). In the Mediterranean, annual mean temperatures on land and in the sea are now approximately 1.5 °C higher than in pre-industrial times and they are projected to rise until 2100 by an additional 3.8-6.5°C for a high greenhouse gas emissions scenario (RCP8.5), and by 0.5-2.0 °C for a scenario (RCP2.6) compatible with the UNFCCC Paris Agreement [START_REF] Abrantes | The climate of the Mediterranean region: research progress and climate change impacts[END_REF], due to both local and global scale drivers [START_REF] Cherif | Climate and Environmental Change in the Mediterranean Basin -Current Situation and Risks for the Future. First Mediterranean Assessment Report[END_REF]. Extreme events are already detectable in different Mediterranean subregions and they are projected to increase in duration, intensity and frequency on land and in the sea, affecting resources and the livelihoods of millions of people living in this region [START_REF] Coma | Global warming-enhanced stratification and mass mortality events in the Mediterranean[END_REF][START_REF] Pausas | Greening and Browning in a Climate Change Hotspot: The Mediterranean Basin[END_REF]. Although the consequences of recent environmental change are threatening all Mediterranean nations, a comprehensive and reliable assessment of the risks has been lacking in impactful scientific reports such as those provided by the Intergovernmental Panel on Biodiversity and Ecosystem Services (IPBES) and the Intergovernmental Panel on Climate Change (IPCC). Consequently, significant efforts are still needed for knowledge transfer from scientists to policymakers in order to make the necessary decisions and implement tangible actions in the Mediterranean [START_REF] Diffenbaugh | Heat stress intensification in the Mediterranean climate change hotspot[END_REF][START_REF] Maiorano | The future of terrestrial mammals in the Mediterranean basin under climate change[END_REF][START_REF] Hare | Climate hotspots: key vulnerable regions, climate change and limits to warming[END_REF][START_REF] De Sherbinin | Climate change hotspots mapping: what have we learned?[END_REF][START_REF] Guiot | Climate change: The 2015 Paris Agreement thresholds and Mediterranean basin ecosystems[END_REF][START_REF] Lionello | The relation between climate change in the Mediterranean region and global warming[END_REF][START_REF] Cramer | Climate change and interconnected risks to sustainable development in the Mediterranean[END_REF][START_REF] Pausas | Greening and Browning in a Climate Change Hotspot: The Mediterranean Basin[END_REF]. Thus, the need to focus on the Mediterranean Basin in the context of recent environmental changes was the core idea behind the 1 st Mediterranean Assessment Report (MAR1), written to provide science-based guidance to multiple actors involved in tailoring a response to climate and environmental changes, and to reduce associated risks for communities and natural ecosystems in the Mediterranean region.

MedECC NETWORK: THE BASELINE OF 1 ST MEDITERRANEAN ASSESSMENT REPORT (MAR1)

The MedECC network was formed in 2015, based on the realization that the shared history and the close connections between countries and regions bordering the Mediterranean, call for strong cooperation with respect to adaptation to, and mitigation of, ongoing environmental change -however, efforts in that direction have been poorly coordinated. Existing assessments cover only parts of the region, some topics and were scattered in various outputs. Substantial scientific knowledge and monitoring data made available by certain research institutions around the Mediterranean was not capitalized on, resulting in an insufficient guidance to policy at regional, national and local levels. The Mediterranean did not follow the course of some other regions and global bodies in producing a relevant scientific basis for policy. Endeavors in institutional coordination and funding led to the establishment of the MedECC network and the launch of scientific work. The MedECC scientific assessment of climate and environmental change is supported by the Union for the Mediterranean, in cooperation with Plan Bleu (UNEP/ MAP Regional Activity Center).

MedECC currently consists of approximately 600 independent scientists from 35 countries working towards a regional science-policy interface for climatic and other environmental changes across the Mediterranean, with clear goals summarized in Figure 1. MedECC's collaborative regional work with a global relevance is embodied through the MedECC vision that valorizes the shared space of peace, development and human rights to better understand climate change and environmental patterns and their effects in the Mediterranean basin to ultimately provide solutions. For this reason, MedECC has been co-awarded the 2020 North-South Prize of the Council of Europe (CoE 2020).

MAR1 assesses current knowledge as it emerges out of the existing body of scientific research. Although it is a regional study, its results may provide useful conclusions on a global scale, as the Mediterranean Basin is always considered as a 'hotspot' [START_REF] Pérès | The Mediterranean benthos[END_REF][START_REF] Garrett | The Mediterranean Sea as a Climate Test Basin[END_REF][START_REF] Giorgi | Climate change projections for the Mediterranean region[END_REF][START_REF] Coll | The Biodiversity of the Mediterranean Sea: Estimates, Patterns, and Threats[END_REF], and a test basin where all global phenomena occur with more remarkable trends [START_REF] Garrett | The Mediterranean Sea as a Climate Test Basin[END_REF][START_REF] Robinson | Mediterranean sea circulation[END_REF]. Hereafter, we summarize the methodology used during the MAR1 drafting, its structure, and its main findings.

METHODOLOGY OF THE MAR1 REPORT

Scoping, Drafting and Review

In order to produce a detailed outline of MAR1, a scoping event was held in 2016, and received the COP22 label. The drafting process of MAR1 (Fig. 3) was a collaborative work that comprises around 190 authors from 25 countries distributed between Coordinating Lead Authors (CLAs), Lead Authors (LAs) and Contributing Authors (CAs) from various Mediterranean sub-regions. CLAs and LAs were selected based on self-nominations in reply to the call by the MedECC Steering Committee (SC). Afterwards, CAs were invited by chapters' CLAs according to the needed expertise to cover the widest possible range of disciplines. The drafting process was based on a compilation of the existing knowledge through a synthetic analysis of mainly peer-reviewed literature and national institutional or government reports and statistics, with some synthetic figures being plotted in the process with no additional research undertaken by the authors. A scientific literature database was maintained by the MedECC Secretariat and was made available to all report authors [START_REF] Lange | Introduction[END_REF].

Since the early stages of the report drafting, chapters have been subject to scientific reviews, allowing suggestions and amendments by scientific experts. A first internal review of the First Order Draft (FOD) involved the SC and the authors of the report, and the Second Order Draft (SOD) was submitted to external scientific reviewers. Moreover, a Summary for Policymakers (SPM) was finally submitted to stakeholders, representing the governments and partner institutions through the Union for the Mediterranean (UfM) and United Nations Environment Programme / Mediterranean Action Plan (UNEP/ MAP) to review the clarity and the coherency of the messages.

Structure and Uncertainties

The report starts with a background about the context and the importance of such assessment efforts (chapter 1), before detailing the main drivers of change in the Mediterranean Basin -which are mainly linked to climate, pollution, land and sea uses, and non-indigenous species (chapter 2), their effects on its resources, water, food, energy (chapter 3), its marine, coastal and terrestrial ecosystems (chapter 4), and society -mainly development, health and human security (chapter 5), while lastly, providing a few case studies and success stories that can be used to manage future risks and improve socio-ecological resilience (chapter 6).

Throughout the report, scientific confidence in its findings is indicated based on the consistency of evidence and the degree of agreement of the scientific community, summarized by the terms "high", "medium" and "low" confidence. Additionally, the report adequately communicates scientific uncertainties, confidence, and the likelihood of outcomes in the material used (see chapter 1; [START_REF] Lange | Introduction[END_REF].

Resources

For water, climate change, in interaction with other drivers (mainly demographic and socio-economic developments), has mainly negative consequences for the water cycle in the Mediterranean Basin, including reduced runoff and groundwater recharge, increased crop water requirements, increased conflicts among users, and increased risk of overexploitation and degradation of water resources. These impacts, leading to water scarcity, are influencing over 180 million people in the South and East countries (high confidence), and will be much more important for global warming, with temperature increases higher than 2°C (Fig. 4; [START_REF] Fader | Water. In Climate and Environmental Change in the Mediterranean Basin -Current Situation and Risks for the Future. First Mediterranean Assessment Report[END_REF]. For food, climate change and extreme events (i.e., heat waves, droughts, floods, sea level rise, etc.) are threatening the food security of Mediterranean nations (high confidence) by causing crop yield losses/failures, crop quality reduction and impacts on livestock. Climate projections show an increasing risk on the food production system in the Mediterranean Basin, such as a 17% decline of crop yields by 2050 (medium confidence) (Fig. 4; [START_REF] Mrabet | Food. In Climate and Environmental Change in the Mediterranean Basin -Current Situation and Risks for the Future. First Mediterranean Assessment Report[END_REF]. Energywise, the report has demonstrated a steady rise of approximately 1.7% each year of the primary energy consumption in the Mediterranean Basin from 1980 to 2016, attributed to a steady increase in the consumption of oil, gas, nuclear and renewables, and caused by changes in demographic, socioeconomic (lifestyle and consumption) and climatic conditions in the region (high confidence) [START_REF] Drobinski | Energy transition in the Mediterranean[END_REF].

Ecosystems

Climate change is affecting all Mediterranean ecosystems (marine, coastal and terrestrial; [START_REF] Balzan | Ecosystems[END_REF]. For marine ecosystems, the results show an increase of species listed as vulnerable (21%) and endangered (11%), increasing signs of "tropicalization" of Mediterranean fauna and flora (medium confidence), and more literature exposing the effects of climate change consequences (i.e., warming, acidification, heat waves, etc.) on all marine trophic chain components, from its primary producers to its marine mammals (high confidence). Projections show that numerous species extinctions would occur, i.e., 40% of endemic fish species will be extinct (Fig. 4) and natural habitats of numerous commercially valuable species will be modified by the end of this century, which would have many repercussions on marine ecosystems' services, such as tourism, fisheries, climate regulation, and ultimately, on human health (medium confidence). For coastal ecosystems, climate change and anthropogenic pressures are impacting various coastal ecosystem regimes (lagoons, deltas, salt marshes, etc.) and coastal infrastructures, affecting thus the flow of nutrients towards the sea, as well as the magnitude, timing and composition of plankton blooms, which significantly increase the number and frequency of jellyfish outbreaks (high confidence), and could have negative impacts on fisheries. Extensive urbanization added to climate and environmental change is also expected to threaten coastal ecosystems, mainly in the southern realm of the Mediterranean (medium confidence). For terrestrial ecosystems, fast and great biodiversity changes have been detectable in the Mediterranean over the past 40 years compared to other areas of the world. On the southern Mediterranean shore, the ecosystems are at risk of fragmentation or disappearance due to human pressure from clearing and cultivation, overexploitation of firewood and overgrazing (high confidence). Drylands are undergoing an overall increase in surface in response to climate change and extensive land abandonment, while 48% of the Mediterranean wetlands were lost between 1970 and 2013, with 36% of wetland dependent animals in the Mediterranean threatened with extinction (medium confidence). Projections indicate negative impacts on terrestrial biodiversity, forest productivity, burned areas [up to a 40% increase for 1.5°C warming, and up to a 100% increase compared to current levels for 3°C warming by the end of the 21 st century (high confidence) ; Fig. 4], freshwater ecosystems and agrosystems with decreased hydrological connectivity, increased concentration of pollutants during droughts, changes in biological communities as a result of harsher environmental conditions, and a decrease of biological processes such as nutrient uptake, primary production, or decomposition.

Society

All the above-mentioned changes are affecting directly and indirectly the human health of Mediterranean populations in different ways, depending on population density, level of economic development, food availability, income level and distribution, local environmental conditions, pre-existing health status, and the quality and availability of public health care (high confidence) [START_REF] Paz | Health. In Climate and Environmental Change in the Mediterranean Basin -Current Situation and Risks for the Future. First Mediterranean Assessment Report[END_REF]. Moreover, climate change and extreme events may have negative impacts on the mental health of people directly affected by consequences, i.e., loss of homes, destruction of settlements and damage to community infrastructure (high confidence). Therefore, sustainable development strategies and innovations (such as climate proofing infrastructures) are key to address the needs of current and future generations, by utilizing natural resources in ways that preserve and sustain them, and ensuring equitable access to those resources in the present and the future (high confidence). However, poverty, inequalities and gender imbalances relate both directly and indirectly to the achievement of sustainable development in Mediterranean countries [START_REF] Santos | Development[END_REF]. All these changes may influence human security, generating additional conflicts and threats linked to water, food, health and others. It is likely that climate change could further exacerbate political instability in the poorest parts of the Mediterranean Basin (medium confidence) [START_REF] Koubi | Human security[END_REF].

Managing future risks and building socio-ecological resilience

Finally, MedECC (2020) sheds light on the importance of managing future risks by highlighting: 1-the current understanding on the trajectory, intensity and spatial extent of future risk for the principal hazards, and associated policy considerations of the region, 2-the current management and adaptation approaches, and prevalent governance frameworks for coping with these risks, 3-a range of examples of adaptation and mitigation for sectorial approaches, while considering case studies from Mediterranean-type environments. A number of innovative and successful practices for achieving sound and sustainable development in countries of the Mediterranean Basin are proposed therein, while highlighting the crucial role of developing joint, region-wide, and integrated management and adaptation approaches that treat multiple hazards in a holistic manner is of utmost importance for sustainable development in the entire region [START_REF] Vafeidis | Managing future risks and building socio-ecological resilience in the Mediterranean[END_REF].

CONCLUSIONS

Based on a synthetic compilation of the existing knowledge in literature, MAR1 highlights the main drivers affecting the Mediterranean Basin, their current effects on resources, ecosystems and society, as well as the projected trends and risks in the context of climate and environmental changes. The outcomes show that more effective policy responses will imply both, strengthened mitigation of the drivers of climate and environmental change such as greenhouse gas emissions, but also enhanced adaptation to impacts. Poverty, inequalities and gender imbalances presently hamper the achievement of sustainable development and climate resilience in Mediterranean countries. Culture is a key factor to the success of adaptation policies in the highly diverse multicultural setting of the Mediterranean Basin. Policies for climate adaptation and environmental resilience potentially infringe on human rights -they need to account for concerns such as justice, equity, poverty alleviation, social inclusion, and redistribution. To support policies for sustainable development with scientific evidence concerning climate and environmental change, a synthesis of current scientific knowledge, covering most relevant disciplines, sectors and sub-regions is presented by the 1 st Mediterranean Assessment Report (MAR1). The findings of MAR1 constitute a foundation for meeting sustainable development needs whilst informing strategic planning and investment at the regional level. The report also provides innovative and successful case studies that would improve the socio-ecological resilience of the different Mediterranean components to resist climate change impacts and ultimately, satisfy the United Nations Sustainable Development Goals (UN SDGs).

Figure 1 :

 1 Figure 1: Goals of MedECC

Figure 2 :

 2 Figure 2: Scientific disciplines of MedECC members

Figure 3 :

 3 Figure 3: The front page of the MAR1

Figure 4 :

 4 Figure 4: The main messages of MAR1 related to risks and impacts on climate and environmental change on the different components of the Mediterranean Basin

ACKNOWLEDGEMENTS

The authors of this paper would like to thank all who have contributed to the MAR1, from coordinators to CLAs, LAs and CAs, as well as all the reviewers and governments' representatives for their comments. Authors are also grateful to all those who have supported MedECC and are all mentioned at: https://www. medecc.org/wp-content/uploads/2020/11/MedECC_MAR1_ Front_matter_ENG.pdf.


Sakonwan Kuhaudomlarp

Eike Siebs

Elena Shanina

Jérémie Topin

Ines Joachim

Priscila Da

Silva Figueiredo

Celestino Gomes

Annabelle Varrot

Didier Rognan

Christoph Rademacher

Anne Imberty
email: anne.imberty@cermav.cnrs.fr

Kuhaudomlarp

Shanina

Ines Topin

Priscila Joachim

Silva Da

Celestino Figueiredo

Gomes

Varrot

Christoph Rognan

Rademacher

Imberty

Titz

A Titz

Non-Carbohydrate Glycomimetics as Inhibitors of Calcium(II)-binding Lectins

Keywords: carbohydrates, catechol, glycomimetic, lectin, PAINS

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Glycoconjugates on the cell surface of host tissues serve as recognition patterns for microbial carbohydrate-binding proteins, lectins, in the initial steps of an infection with a pathogen. [1] The co-evolution between host glycoconjugates and pathogen receptors resulted in the ability of lectins to decipher the three-dimensional structure of complex branched oligosaccharides, referred to as "glycocode". [2] Among pathogens, Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium inter alia responsible for pneumonia in immunocompromised patients, those suffering from cystic fibrosis (CF), or patients under ventilation. [3] P. aeruginosa starts forming a biofilm on its colonization site to shield the bacterium from antimicrobial treatment and the immune response and thus hampers combating the bacteria. Host recognition, tissue adhesion and biofilm formation of P. aeruginosa are mediated by two lectins: d-galactose-specific and l-fucose/d-mannose-specific LecA and LecB, respectively. [4] Both lectins are virulence factors and have been considered as targets for the development of carbohydratebased anti-infective compounds and for the delivery of antibiotics via carbohydrate targeting moieties. [5] LecA is of special interest because of its ability to damage cell membranes and to facilitate bacterial cell internalization. [START_REF] Eierhoff | Proc. Natl. Acad. Sci[END_REF] LecA is a homotetramer containing one calcium ion in the carbohydrate-binding site coordinating amino acids and galactosides (Figure 1 A,B). [START_REF] Cioci | [END_REF] The target of this lectin is likely the globotriaosylceramide glycolipid that presents the Gala1-4Gal epitope. [8] The relatively low affinity for galactose or galactobiose (K d = 50 mM) is overcome by high avidity with divalent or multivalent ligands that can cross-link the neighbouring binding sites (K d = 1-20 nM). [5a, 9] However, because of the presence of a hydrophobic surface nearby the galactose-binding site, monovalent galactosides with aromatic aglycons demonstrated high affinity for the lectin in the low micromolar range. [10] Recently, we reported an epoxide-carrying galactoside that binds covalently to a cysteine residue present in the carbohydrate-binding site of LecA. [11] Targeting LecA is a promising way to counteract P. aeruginosa infections. The chemical properties of the binding pocket of LecA were used as an inspiration for the synthesis of many compounds, all maintaining the presence of a galactose residue for the primary calcium-dependent binding site. [5a,e, 12] The development of non-carbohydrate mimics would be an alternative strategy, opening a large chemical space for novel inhibitors. Indeed, since carbohydrate-protein interactions are mostly governed by a complex arrangement of hydrogen bonds and hydrophobic contacts, the success of non-carbohydrate analogues is challenging. Non-carbohydrate glycomimetics were obtained for the model plant lectin Concanavalin A [13] and virtual screening resulted in ligands with higher affinities than the natural ligand mannose. [14] Apart from this work on a model lectin, only C-type lectins, that is, calcium-dependent animal lectins with roles in inflammation and immunity, have been targeted. Several compounds for the selectins have been obtained. [12,15] Glyco-mimetics based on a shikimic acid scaffold have shown good inhibitory activity against the C-type lectin DC-SIGN. [16] The presence of a calcium ion is therefore a promising factor for a glycomimetic strategy against LecA.

Virtual screening is a powerful technique to identify potential ligands from large chemical databases. [17] Therefore, this approach was applied to screen the National Cancer Institute (NCI) Diversity IV database for identifying noncarbohydrate ligands directed to the galactose binding site of LecA. The best virtual screening hits were experimentally tested in a competitive binding assay based on fluorescence polarization, [18] identifying catechol-containing compounds as binder. Further, catechols were confirmed in orthogonal biophysical methods: a thermal shift assay (TSA), ligand-and protein-observed 19 F (PrOF) and 1 H-15 N TROSY NMR spectroscopy, surface plasmon resonance (SPR) and X-ray crystallography was employed to elucidate their binding modes to LecA. This work demonstrates the first example of a novel group of non-carbohydrate glycomimetic compounds targeting a bacterial lectin, which can be subsequently developed into a novel class of LecA inhibitors. 2.

Results and Discussion

Virtual Screening of Ligands for LecA from NCI and Validation of Catechol-Containing Hits in a Competitive Binding Assay A docking protocol was designed using Glide [19] and the 1.5 resolution crystal structure of LecA in complex with a galactoside (PDB code 3ZYH). [10a] After internal validation with ligands of known structures and affinities, the E-model score function was selected for ranking of orientations together with MM-GBSA [20] for ranking of energies (Figure S1). In total 1597 molecules from the National Cancer Institute (NCI) diversity set IV were docked into the binding site of LecA. Only those poses presenting a minimum of four contacts in the site, that is, hydrogen bonds and coordination to the calcium ion, were retained and ranked based on MM-GBSA rescoring function which resulted in the top 60 poses (Table S1). Those poses with at least two contacts to the calcium ion were manually clustered according to their similarity in binding mode into 15 groups and ranked as a function of their lower energy pose. We identified the 10 top clusters shown in Figure S2 that illustrate the encountered variety of chemical space. Interestingly, clusters 1, 5 and 6 corresponded to analogs of nucleotides, binding to LecA through 5-membered furanose rings. Other molecules bound to the calcium ion through glycerol side chains (cluster 3), sulfate groups (cluster 4) or a carboxylate (cluster 9). Of special interest was cluster 2 with five molecules (Figure 1 C) that have a catechol motif to coordinate to the calcium ion with the two vicinal oxygen atoms in a very conserved geometry (Figure 1 D). Finally, the lowest energy hit, 4isobutyryl catechol (compound b in Figure 1 C, 21 in Table 2), allows coordination to the calcium ion and an appropriate balance of hydrogen bonds and hydrophobic contacts that mimics the binding of galactose (see Figure S3).

Initially, 40 compounds derived from virtual screening were selected based on structural diversity (Table S1), purchased and screened in the established competitive binding assay based on fluorescence polarization at a single concentration of 3.3 mM. Among the 40 compounds tested, 4isobutyryl catechol (b in Figure 1 C, compound 21 in Table 2) was identified as weak inhibitor (approx. 10 % inhibition at 3.3 mM). It was the only molecule able to displace the fluorescent galactoside ligand in this assay. Therefore, both virtual and experimental screening suggest the catechol as a new scaffold for LecA.

Experimental Screening of a Catechol Library

Despite the fact that some catechols are approved drugs, for example, the b 1 -adrenergic agonist dobutamine or the antibiotic cefiderocol, this class of compounds is well known as frequent hitter and pan-assay interference compounds (PAINS). [21] Catechols are prone to oxidation into highly reactive quinones, which are reactive towards nucleophiles and may lead to an unspecific interaction with proteins. [22] However, the concept of a general exclusion of PAINS has been recently questioned based on extensive analysis of publicly available assay data [23] and a case-to-case consideration using orthogonal assay activity has been stated necessary.

The structure-activity relationship of catechols was followed up by purchasing 29 further commercial catechols with different substitution patterns and biophysical analysis using various orthogonal methods (Table 1, Table 2). Unsubstituted All compounds were tested in a modified competitive binding assay with a Cy5 dye conjugated to a galactoside S7 (synthesis of S7 see Scheme S1, binding of S7 to LecA see Figure S4). This change in fluorescent dye was necessary to circumvent spectral overlap of some catechols with the previously used fluorescein conjugate. [18] After 1 h incubation, various catechols bearing electron-withdrawing substituents showed a weak inhibition of LecA. Surprisingly, the polarization measured for some of the tested compounds has changed dramatically after extended incubation (16 h). While some catechols showed a constant inhibition over time, in particular catechols bearing electron-donating substituents such as tert-butyl or methyl groups showed the strongest increase in inhibition.

To avoid the false-positive detection of an apparent inhibition due to an unspecific reactivity of potentially formed quinones, 2-mercaptoethanol was added to the competitive binding assay as an agent to trap those reactive species at equimolar concentration to the highest catechol concentration. Interestingly, the previously active electron-rich catechols were inactive under these conditions (Figure S5). In contrast, LecA inhibition of catechols carrying electron withdrawing substituents, such as nitrile 3, nitro 12, and ketone 21, has not changed in presence of 2-mercaptoethanol and over time (Table 1, Table 2).

At a concentration of 4 mM, ten out of the 30 catechols tested showed a quantifiable and 2-mercaptoethanol-independent inhibition of LecA in this competitive binding assay (Table 1, Table 2, Figure 2 A). Benzoylcatechol 23 was the most active compound with an inhibition of 39 %. Docking of 23 into the LecA binding site suggests that the benzoyl ring can interact with Gln53 (Figure S3A). In the competitive binding assay, this compounds activity is followed by nitrocatechol 12 and cyanocatechol 3 with inhibition of 33 % and 23 %, respectively. All three compounds were two-to fourfold more active than the initial in silico hit, isobutyryl catechol 21 (11 % inhibition). Even though some of the catechols, such as 4-parachlorobenzoyl catechol (24), were only soluble at the test concentration of 4 mM in presence of 25 % DMSO as a cosolvent, LecA bound successfully to the Cy5 galactoside S7 and 24 showed 22 % inhibition of LecA. These results support the notion that electron-deficient catechols can either allosterically inhibit the LecA-galactoside interaction or competitively bind to its carbohydrate-binding site.

Secondary Assays Verifying Binding of Catechols to LecA.

First, a subset of electron-rich and electron-deficient catechols was tested in a thermal shift assay to verify their interaction with LecA. In case of a ligand binding event, the thermal stability of a protein increases due to stabilizing interactions in the ligated state. [24] Therefore, the melting point of LecA was measured in absence of ligand (T m 89.4 8C), in presence of a galactoside (T m 91.1 8C) or thirteen catechols (Figure S6).

A general trend could be observed where the electrondeficient catechols, that is, nitriles 3 and 9, acetyl 17, isobutyryl 21 and benzoyl 23, led to an increase of the melting point of LecA. In particular, 4-benzoyl catechol 23 and 3- cyano catechol 3 had the largest effect on the melting point of LecA with an increase of + 0.4 8C and + 1.8 8C, respectively (Tables 1,2, Figure S6). In contrast, the electron-rich catechols (methyl 11 and tert-butyl 13) and the unsubstituted catechol 1 destabilized LecA and thus, lowered its melting temperature. A strong destabilization was observed in case of 4-tert-butyl catechol 13 (À15.4 8C) and 4-methyl catechol 11 (À11.0 8C).

As an orthogonal binding assay ligand-observed NMR spectroscopy experiments monitoring the T 11 relaxation of the electron-deficient catechol, virtual screening hit isobutyryl 21, and the electron-rich catechol 4-tert-butyl catechol 13 were measured. The T 11 relaxation rates of a small molecule depend on its correlation time and hence can be used to detect binding to higher molecular weight receptors. [25] Therefore, T 11 relaxation of isobutyryl catechol 21 or tertbutyl catechol 13 were measured in presence and absence of LecA, and both compounds showed an increase in relaxation with LecA (Figure 2 B, Figure S7). However, the relaxation rate of isobutyryl catechol 21 was strongly increased in presence of LecA (red) and could be fully reversed to the free state in presence of an excess of Me-a-d-Gal (blue). The effect of tert-butyl catechol 13 with LecA was much weaker and addition of Me-a-d-Gal had no significant effect. Since the binding of isobutyryl catechol 21 to LecA can be fully competed with a galactoside, this data suggests that electrondeficient catechols bind LecA in the carbohydrate-binding site. Moreover, this result is in agreement with our competitive binding assay with a fluorescent tracer, where isobutyryl catechol 21 inhibited LecA and the inhibition of tert-butyl catechol 13 was unspecific and absent in presence of a scavenging nucleophile.

Both assays, T 11 relaxation NMR spectroscopy and the thermal shift assay, have further validated electron-deficient catechols as ligands of LecA and NMR gave strong hints for a direct galactose-competition as mode of action. Next, we quantified the catechols binding affinity using surface plasmon resonance (SPR) as an orthogonal method and localized their binding site by protein-observed NMR spectroscopy and X-ray crystallography.

Quantification of direct catechol binding to LecA was performed using SPR analysis. Binding analyses were performed by injecting various catechols at two concentrations (0.2 and 1 mM, except for 16 and 27 which were injected at 0.2 and 0.5 mM). We identified 14 catechol hits by SPR, six of which were in agreement with other biophysical assays (nitrile 3, hydroxymethyl 6, nitro 12, acetyl 17, benzoyl 23 and dihydroxybenzoyl 25, Figure 3 A, Figure S8). Subsequently, multicycle kinetic analyses were performed on these six prioritized hits to obtain the corresponding dissociation constants (K d ) (Figure 3 B, Figure S9). These data showed that hydroxymethyl 6, acetyl 17 and dihydroxybenzoyl 25 have very weak affinity towards LecA (extrapolated K d of > 100 M) (Figure S9), whereas compounds nitrile 3, nitro 12 and benzoyl 23 catechols have promising K d values in the low millimolar range (Figure 3). The lowest K d value was obtained for nitro 12 (K d = 0.56 AE 0.34 mM) followed by nitrile 3 (K d = 1.11 AE 0.07 mM) and the most active compound in the competitive binding assay, benzoyl 23, was somewhat less active (K d = 3.46 AE 0.41 mM). Finally, the initial virtual screening hit isobutyryl 21 had a K d of approx. 145 M corresponding to the results from the competitive binding assay. For weaker binding hits, the efficiency was used for comparison of the affinity, among which 26 showed the highest efficiency (142.6 %), which is suspiciously high for a small molecule. We hypothesized that this unexpected high binding response observed on SPR is due to a potential oxidation and unspecific interaction with the protein.

Protein-Observed NMR Reveals Catechols Targeting the Carbohydrate-Binding Site of LecA

Protein-observed 19 F (PrOF) NMR spectroscopy is a sensitive technique to spot weak binders in a low millimolar affinity range. Here, we used previously established PrOF NMR with 5-fluorotryptophan-labeled (5FW)-LecA [26] to determine the binding region of catechols. First, we measured 5FW-LecA in presence of para-nitrophenyl b-d-galactoside (pNPGal) as well-known ligand for the carbohydrate-binding site of LecA. As expected, the resonance of the 5-fluorotryptophan located in the carbohydrate-binding site, W42, showed a clear chemical shift perturbation (CSP) towards a ligand-bound form in presence of pNPGal indicating that PrOF NMR can be used to spot the binding site of ligands (Figure 4).

Next, we validated 30 catechols in PrOF NMR (Table S2) and observed the perturbation of W42 in presence of 18 catechols that target the carbohydrate-binding site (Figure S10). Notably, the chemical shift perturbation of W42 in presence of the electron-deficient benzoyl catechol 23 was comparable to pNPGal (Figure S11) and nitrile 3 also induced a chemical shift perturbation of the W42 resonance (Figure 4).

We further analyzed uniformly 15 N-labelled LecA in 1 H, 15 N TROSY NMR experiments in presence and absence of two galactosides or four catechols (Figure 4). The fingerprint of the resulting chemical shift perturbations was highly similar for d-Gal and pNPGal compared to the three tested catechols 3, 23 and 25 (Figure 4).

Taken together, compounds nitrile 3 and benzoyl 23 show a comparable binding of LecA in protein-observed NMR spectroscopy as the positive control pNPGal allowing us to hypothesize that both compounds target the carbohydratebinding site of LecA.

X-Ray Crystallography Reveals Glycomimetic Binding Mode of One Catechol Hit

To further investigate the interaction between the catechol hits and LecA at atomic resolution, we co-crystallized LecA with the six hits that were identified among all biophysical assays, nitrile 3, hydroxymethyl 6, nitro 12, acetyl 17, benzoyl 23 and dihydroxybenzoyl 25. We adapted a dry cocrystallization approach previously reported by Gelin et al., which involves deposition of the compound onto the crystallization slide and gentle evaporation of DMSO prior to dispensing the protein onto the dried compound. [27] This approach enables the deposition of high amounts of the dried compounds onto the slide, whilst avoiding complicated crystal manipulation and use of high DMSO concentrations. Protein crystals were obtained for co-crystallization of LecA with nitrile 3, benzoyl 23 and dihydroxybenzoyl 25. X-ray diffraction data were collected from crystals of LecA in complex with nitrile 3 (LecA-3) and in complex with dihydroxybenzoyl 25 (LecA-25) whereas the crystals of LecA with benzoyl 23 diffracted poorly (resolution > 4 ) and were not further investigated. Both complex structures were successfully solved at 1.84 in space group I 2 (for LecA-3) and P2 1 22 1 (for LecA-25) (Table S3).

The overall structure of LecA-25 is very similar to LecAgalactose (global RMSD = 0.29 ). Intriguingly, the electron density corresponding to compound 25 was found remotely from the galactose binding site (Figure S12A), which was unexpected considering its ability to outcompete the galactose-derived fluorescent probe. We hypothesized that the interaction of 25 with the remote site on LecA maybe a crystallization artefact since the molecule locates at the interface of the protein crystal contacts and forms only minimal interactions with the protein (Figure S12B).

The LecA-3 structure is also very similar to the LecAgalactose complex (PDB: 1OKO) with a global RMSD of 0.8 (superposition of equivalent Ca atoms). Here, however, catechol 3 occupies the previously reported galactose binding site in monomers A and C of the LecA tetramer (Figure 5 A andB). The two vicinal hydroxy groups of 3 coordinate the Ca 2+ ion and form hydrogen-bonded interactions with the side chains of Asp100 and Asn107 and the backbone oxygen of Tyr36 (Figure 5 D), the interaction of which imitates the roles of OH3 and OH4 of galactose in the LecA-galactose interaction (Figure 5 C). A conserved water molecule (WAT1) forms hydrogen bond bridges between the nitrogen atom of the nitrile in catechol 3 and the oxygen atom the carbonyl group of Pro51 (Figure 5 D). The nitrogen atom thus assumes the role of OH6 in the LecA-galactose interaction, where hydrogen bond bridges through WAT1 were identified between the OH6 group and Pro51 (Figure 5 C). Shifting the nitrile group to the neighboring carbon atom, that is, to position R2 in compound 9 instead of position R1 in compound 3, resulted in approximately a threefold-reduction of the SPR efficiency and inhibition in the FP assay (Table 1), which further substantiates the essence of the positioning and the role of the nitrile group in the LecA-3 interaction.

To investigate if catechols interact with other Ca 2+binding lectins and could be used as a general motif for glycomimetics, we performed 1 H-15 N HSQC NMR with 15 Nlabeled Langerin CRD (Figure 6 A). We observed that 3 promoted chemical shift perturbations (CSPs) of amino acids located in the carbohydrate-binding site of Langerin (Figure 6 B), but also affected amino acids that are located at a certain distance. The magnitude of CSPs observed for 3 (tested at 1 mM due to solubility restrictions) was generally weaker than mannose which was tested at 30 mM. Given the allosteric nature of some calcium binding lectins such as Langerin, [16a, 28] changes in the resonances located far away from the carbohydrate-binding site can be correlated to changes in the binding site. Potentially, 3 can be located in the binding site, or in a remote site that triggers the allosteric network.

To demonstrate that 3 targets the carbohydrate-binding site of Langerin, we performed 1 H STD competition NMR experiments (Figure 6 C). For this, we recorded STD spectra of 3 without or with Langerin ECD and upon addition of mannose as the competitor. As result, we observed a 65 % reduction in peak intensities of resonances from compound 3 in STD NMR spectra in presence of mannose indicating its partial competition from the carbohydrate-binding. This is in agreement with 1 H-15 N HSQC data, where 3 has been observed to alter resonances from both the carbohydrate-and secondary binding sites in Langerin. Thus, these data reveal catechol 3 as a general glycomimetic motif for a bacterial and a mammalian calcium-binding lectin.

Conclusion

In this work, a non-carbohydrate glycomimetic has been identified for a relevant anti-infective drug target. We have established catechols as mimicry of the carbohydrate ligand, binding to the protein via the crucial calcium-ion present in the carbohydrate-binding site of LecA, a key lectin involved in infection mechanisms of the ESKAPE pathogen P. aeruginosa. Therefore, the catechols have good potential beyond LecA and may also serve as glycomimetics for the large family of animal C-type lectins and other bacterial lectins relying on calcium-mediated glycan recognition. Target specificity required for a selective drug can be achieved by further optimizing a central catechol motif addressing the carbohydrate-binding site.

Despite the widespread belief, catechols are not necessarily PAINS when care is taken in analysing their binding properties in orthogonal assays under appropriate experimental conditions. We showed that electron-deficient catechols are stable under the conditions tested and their interaction with LecA has been confirmed in several orthogonal assays. In contrast, electron-rich catechols proved to be unspecific binders and their observed effect on LecA could be reversed in presence of a competing nucleophile, 2-mercaptoethanol. From an initial virtual screening approach for the bacterial lectin LecA, we have identified electron-deficient catechols as non-carbohydrate inhibitors for the lectin LecA. We validated our hits in various orthogonal biophysical assays. The binding of the initial catechol hit isobutyryl 21 derived from virtual screening was confirmed in a competitive binding assay based on fluorescence polarization. This assay and a thermal shift assay were used to demonstrate that only catechols with electron-withdrawing groups are robust inhibitors of LecA, while catechols with electron-donating substituents showed protein destabilization and unspecific inhibition which was absent in presence of a nucleophilic scavenger. This specificity of catechols was further confirmed in ligand-observed T 11 -relaxation NMR experiments for electron-deficient isobutyryl catechol 21 and electron-rich 4tert-butylcatechol 13. The binding affinity of catechols was determined by SPR and led to K d -values in the millimolar range, for example, nitrile 3 with a K d of 1.11 AE 0.07 mM and nitro 12 with a K d of 0.56 AE 0.34 mM. These affinities of the fragment hits (MW 135-155 g mol À1 ) result in ligand efficiencies of 0.40 for both compounds, providing a good basis for future fragment growing. 19 F-protein observed NMR spectroscopy revealed that the catechols with electron-withdrawing substituents were binding to the carbohydrate-binding site of LecA. Finally, X-ray analysis of LecA in complex with nitrile 3 unambiguously confirmed catechols as galactosemimicking ligands located in the carbohydrate-binding site. The structure revealed that catechols were coordinating to the calcium ion via their vicinal hydroxy groups, and the nitrile group underwent a hydrogen-bonding interaction with a crystal bound water molecule, highly homologous to the primary hydroxy group of galactose in complex with LecA.

This work sets the basis for future optimization of the catechol scaffold to increase binding potency and drug-like properties of a possible antimicrobial agent. Therefore, this work sets a new starting point for the development of future S4) were used for mapping the binding site of 3 on a structure of Langerin CRD (PDB: 3P5D). K257 and K299 are highlighted in the CSP plot as 1 and 87, respectively. C) The competition 1 H STD NMR experiment with mannose was performed with 3 in presence and absence of Langerin ECD. Binding of 3 to the carbohydrate-binding site of Langerin has been confirmed as indicated by decreasing peak intensities in the STD NMR spectrum upon addition of mannose.

LecA inhibitors against biofilm-associated P. aeruginosa infections by developing new non-carbohydrate LecA glycomimetics and provides a structure-based rationale for the development of lectin-inhibitors in general.

Moreover, catechol 3 proved to target the carbohydratebinding site of both Ca 2+ -binding bacterial (LecA) and mammalian (Langerin) lectins. Given that Ca 2+ -binding lectins have been considered "undruggable" for a long time, [29] we believe catechols challenge this paradigm as a novel class of non-carbohydrate glycomimetics. Further growing of this low affinity fragment hit, catechol 3, into lead structures will provide high target affinity. Structure-based design of additional functional groups will provide the specificity towards the lectin of interest for the desired indication.

Figure 1 .

 1 Figure 1. A) Crystal structure of the homotetrameric LecA in complex with galactose (PDB code: 1OKO). B) View of calcium-ion dependent galactose coordination in the carbohydrate-binding site of LecA. C/D) Shown are docking poses of five catechol-containing compounds from the 60 best virtual screening hits. Compound b is identical to 21 in Table2.

Figure 2 .

 2 Figure 2. Analysis of catechol binding to LecA using: A) a competitive binding assay based on fluorescence polarization with catechols 3, 12, 17, 21, 23 and 25 and methyl a-d-galactoside as positive control; B) T11 relaxation NMR spectroscopy of isobutyryl catechol 21 and tertbutyl catechol 13. Signal intensity decay of methyl protons plotted for 21 and 13 vs. spin-lock times.

Figure 3 .

 3 Figure 3. SPR analysis of catechol direct binding to LecA. A) The binding responses at steady state on the sensorgrams, when 0.2, 0.5 or 1 mM of catechols were injected (blue, orange and black dots, respectively), were plotted against injection cycles of compounds. Dose-response increases more than twofold are indicated by dotted circles. The catechol entries corresponding to the dose-response binding are indicated. Positive control (0.1 mM pNPGal represented by crosses) was injected to monitor the activity of immobilized LecA and enable data normalization. B) Multi-cycle kinetic analyses of the prioritized hits nitrile 3, nitro 12, benzoyl 23 by SPR. Left: Sensorgrams, Right: affinity analyses based on the data from the sensorgrams.

Figure 4 .

 4 Figure 4. (left) Protein-observed 19 F (PrOF) NMR spectroscopy of nitrile 3 and pNPGal with 5FW-LecA; (right) 1 H, 15 N TROSY-HSQC NMR spectra (upper panel) of uniformly 15 N-labelled LecA in absence and presence of ligands d-Gal, pNPGal or nitrile 3 and a plot with a 1:0 barcode (bottom panel) shows CSPs and changes in peak intensity of arbitrarily numbered resonances in LecA observed for ligands d-Gal, pNPGal, nitriles 3, 9, benzoyl 23 and dihydroxybenzoyl 25. This data indicated catechols 3, 9, 23, 25 bind LecA in a region similar to d-Gal and pNPGal suggesting binding to the carbohydrate-binding site of LecA.

Figure 5 .

 5 Figure 5. A) Overall structure of LecA-3 complex (PDB code: 6YO3). 2 molecules of compound 3 are shown as sticks and the Ca 2+ ions as green spheres. Dotted circles indicate the binding sites of 3. Each LecA monomer is depicted as cartoon in different colors. B) Surface display of compound 3 at the binding site. C) Interaction of galactose with LecA (PDB code: 1OKO). D) Interaction of 3 with LecA, electron density is displayed at 1s.

Figure 6 .

 6 Figure 6. A) 1 H-15 N HSQC NMR spectra of uniformly 15 N-labelled human Langerin CRD in absence of ligands (grey) and in presence of mannose (red) or catechol 3 (blue). The two resonances K257 and K299 are magnified and shown as an example for the interaction of 3 (blue) with Langerin, which is in the similar manner to mannose (red). B) The perturbed resonances in the CSP plot (CSP > 0.005 p.p.m., for correlation of resonance ID and amino acid see TableS4) were used for mapping the binding site of 3 on a structure of Langerin CRD (PDB: 3P5D). K257 and K299 are highlighted in the CSP plot as 1 and 87, respectively. C) The competition 1 H STD NMR experiment with mannose was performed with 3 in presence and absence of Langerin ECD. Binding of 3 to the carbohydrate-binding site of Langerin has been confirmed as indicated by decreasing peak intensities in the STD NMR spectrum upon addition of mannose.

Table 1 :

 1 Catechols

	FP	FP with 2ME	TSA	SPR	PrOF NMR

and LecA binding by fluorescence polarization (FP), thermal shift assay (TSA), and surface plasmon resonance (SPR). H E-CHC(CN)CONHBn inconcl.* inconcl.* -15.8 at 0.5 mM n.s. Protein-observed 19 F (PrOF) NMR spectroscopy was performed with 5FW-LecA. FP averages and std. dev. from 3 experiments, TSA averages from two experiments, * = tested at 25 % DMSO, 2ME = 2-mercaptoethanol, CSP = chemical shift perturbation, n.s. = not significant, std. dev. < 0.03 ppm.

catechol 1 and various derivatives carrying electron-donating or -withdrawing substituents in different positions were obtained (2-30) among which are fourteen acylated catechols (17-30, including the hit 4-isobutyryl catechol 21).

Table 2 :

 2 Acylated catechols and LecA binding by fluorescence polarization (FP), thermal shift assay (TSA), and surface plasmon resonance (SPR). PrOF) NMR spectroscopy was performed with 5FW-LecA. FP averages and std. dev. from 3 experiments, TSA averages from two experiments, * = tested at 25 % DMSO, 2ME = 2-mercaptoethanol, CSP = chemical shift perturbation, n.s. = not significant, std. dev. < 0.03 ppm.

	FP	FP with 2ME	TSA	SPR	PrOF NMR

Angew. Chem. Int. Ed. 2021, 60, 2 -13 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH www.angewandte.org

The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH Angew. Chem. Int. Ed.

2021, 60, 2 -13

Acknowledgements

The work was supported by the ANR/DFG French-German project (ANR-AAPG-2017). The authors acknowledge financial support from Agence Nationale de la Recherche (grant no. ANR-17-CE11-0048) and from Deutsche Forschungsgemeinschaft (grant no. Ti756/5-1 and RA1944/7-1). J.T. was supported by Labex Arcane/ CBH-EUR-GS (ANR-17-EURE-0003) and we also thank Glyco@Alps (ANR-15-IDEX02) for support. We acknowledge synchrotron SOLEIL (Saint Aubin, France) for access and technical support at beamline PROXIMA1. We thank Emilie Gillon (CERMAV) for providing recombinant LecA for SPR and X-ray crystallographic experiments and Dr. Alberto Plaza (HIPS) for implementing the T11 pulse sequence provided by Dr. Marcel J. J. Blommers (Saverna Pharmaceuticals, Switzerland). Open access funding enabled and organized by Projekt DEAL.

Conflict of interest

The authors declare no conflict of interest.


R Eyssette

F Heymes

A M Birk

Ground loading from BLEVE through small scale experiments: Experiments and results

Keywords:

The ground force generated by a BLEVE is a hazard that has been seldom studied, even though BLEVE has been source of many research works in the past decades. However, emergency responders have been asking questions the risk and consequences of a BLEVE on a bridge and other critical infrastructures for a while, with no answer so far. Moreover, a tank truck accident in Bologna in August 2018 has shown that bridge collapse may result from such scenario.

This paper presents the experimental work done with a small scale apparatus reproducing realistic BLEVE failure with a cylindrical tube. Load cells were placed under the base plate holding the tube to measure the local ground force generated by the explosion. Forces from 10 kN to 55 kN were measured for a 50 mm diameter tube with 300 mm length failing at pressures from 10 to 32 Bar. The ground force signals are interpreted together with the imaging and internal pressure signal. The synchronization between violent boiling, repressurization in the vessel and strong ground force is clear through this comparison. Different ground force signals were observed depending on the fill level and weakened length of the tube. The maximum ground force and impulse vary almost linearly with failure pressure and liquid fill level. It is less clear for the influence of weakened/ opening length of the tube. More data is required to conclude on this parameter influence.

Introduction

BLEVE (Boiling Liquid Expanding Vapor Explosion) incidents have happened around the world for more than 60 years now, on industrial sites as well as on the roads and rails during transportation of pressure liquefied gases in pressure vessels. This type of incident is feared because of the dramatic consequences it often induces. The deadliest case of BLEVE, which happened in Mexico in 1984, took over 650 lives, led to over 6000 injuries, without mentioning the tremendous material cost of the incident [START_REF] Pietersen | The LPG disaster in Mexico[END_REF]. Even though not all BLEVEs are as deadly, they are dangerous and feared by the industries using pressure liquefied gas storage vessels. The material consequences are also often costly. It is thus of major importance to understand the hazards of such an event, to prevent its dramatic consequences and react better when facing an upcoming BLEVE.

A BLEVE is a physical explosion due to vapour release and violent boiling of the superheated liquid. The hazards from a BLEVE include:

• Overpressure from a lead shock wave. This shock wave is argued in recent work to be generated by the vapour space in the vessel [START_REF] Eyssette | Characterization and Modeling of Near-field BLEVE Overpressure and Ground Loading Hazards[END_REF]. • Vessel fragment propulsion from the expansion energy of the whole fluid in the vessel. These fragments are far reaching with trajectories difficult to predict. • Potential fireball or vapour cloud explosion if the content of the vessel is flammable • Toxic cloud dispersion if the content is toxic • Blast wind and dynamic pressure from the two-phase flow following the boiling process. This is arguably taking its source in the liquid boiling and expansion. • The local ground loading due to the content release Many prediction models have been established on several of these hazards. A good review work on the history of BLEVE and the prediction of its various hazards has been done a decade ago [START_REF] Vigilfuoco | BOLOGNA (BO) Loc. Borgo Panigale -Esplosione autocisterna[END_REF]. [START_REF] Abbasi | The boiling liquid expanding vapour explosion (BLEVE): mechanism, consequence assessment, management[END_REF]. The ground force hazard is however not studied so far in the case of BLEVE.

Understanding the physics of BLEVE phenomenon is the starting point of most of these models. A common assumption in many of these models is the use of the liquid energy to estimate the overpressure of a shock. A few references argue that it is a wrong approach to modeling the overpressure, as it seems to be driven only by the vapour phase expansion [START_REF] Baker | Explosion Hazards and Evaluation[END_REF][START_REF] Birk | Near-field BLEVE overpressure effects: the shock start model[END_REF]. However the liquid should still be considered to the hazardous behavior of BLEVE. Indeed most of the thermal energy is stored in the liquid phase, due to its larger enthalpy compared to vapour phase. Thus the boiling process is largely responsible in the fragment propelling hazard, as well as the dynamic loading of surrounding structures and ground loading.

As mentioned above, very little work exists on the prediction of the force generated on the ground when a vessel is subjected to a BLEVE, even though various situations require its investigation. If the vessel is held above ground by a structure on an industrial site, chances are that the structure will not resist the explosion, and structures and objects under the vessel will be destroyed, or structures around the vessel may be pulled down. Emergency responders have also been asking for some years about the consequences of a BLEVE from a tanker truck accident on a bridge: could it destroy a bridge if such incident happened?

A recent incident gave an answer to this question for one specific case. In August 2018, a BLEVE incident happened in Italy: a road tanker truck was involved in a road accident on a highway bridge in the surroundings of Bologna Italy. Two trucks following each other collided. One of them was loaded with liquid propane, the other one was carrying various flammable chemical products. Various flammable liquids were spilled. A fire started in the chemical products transport trailer. Once the fire developed, after a certain time, a very large explosion occurred. It was a blatant case of BLEVE. The vessel was found flattened, and part of the bridge under the truck collapsed after the explosion (Fig. 1). This incident confirmed that the matter needs to be addressed.

Bridges subject to explosions have been studied in literature, with simulations for different bridge configurations [START_REF] Foglar | Full-scale experimental testing of the blast resistance of HPFRC and UHPFRC bridge decks[END_REF][START_REF] Hashemi | Dynamic response of cable-stayed bridge under blast load[END_REF][START_REF] Winget | Analysis and design of critical bridges subjected to blast loads[END_REF], feedback from real accidental cases [START_REF] Wang | Analysis of a bridge collapsed by an accidental blast loads[END_REF] and experiments on smaller scale concrete blocks [START_REF] Wang | Experimental study and numerical simulation of the damage mode of a square reinforced concrete slab under close-in explosion[END_REF]. For most of these studies, the load on the bridge from the explosion is found through the TNT-equivalent model to estimate the overpressure generated in Fig. 2. Characteristic pressure history for venting of R-22 and pressurized water [START_REF] Barbone | Explosive Boiling of a Depressurized Volatile Liquid[END_REF].

the explosion. The ground force hazard is however not studied so far in the case of BLEVE.

Rapid boiling during a BLEVE may produce a significant thrust effect, but little data can be found on the resulting force. Pipe ruptures of superheated liquids were studied by [START_REF] Baum | The thrust exerted on a gas-pressurized vessel during axial rupture[END_REF] and showed the damage involved with the thrust from superheat boiling. These models assume a steady state propagation of the crack along the pipe. Other configurations were studied by the same author, either vertical cylindrical vessel failing at its top, propelling the end cap [START_REF] Baum | Rupture of a gas-pressurized cylindrical vessel: the velocity of a detached end-cap[END_REF], or failing at the bottom [START_REF] Baum | Rocket missiles generated by failure of a high pressure liquid storage vessel[END_REF]. Further investigation of the BLEVE physics in the horizontal cylinder configuration is required to understand its impact on the ground.

Since rapid boiling during a BLEVE is expected to contribute to a dynamic pressure load, several studies investigated transients of pressure while vessels and cylinders open with pressure liquefied gas containment, emphasizing the pressurization process happening due to the superheat boiling inside the container [START_REF] Barbone | Explosive Boiling of a Depressurized Volatile Liquid[END_REF]. Fig. 2 shows the difference of pressure between a superheated liquid venting (refrigerant R-22) and venting with no superheat (compressed air and water) in a rectangular vessel of 260 mL bursting on its top (2 cm wide x19 cm high x7.5 cm deep). Pressure is measured near the opening of the vessel. The pressure drop (quantified by P drop ) is the result of the fluid expansion. In the case of superheated R-22, the boiling process is visible through a pressure rise ( P rise ) preventing the pressure to drop down to atmospheric conditions. This pressure rise shows the intensity of the boiling process. It must be noted that the pressure remains almost constant for some time after the pressure rise in the vessel and then it decays down to ambient pressure. This is due to the restricted cross section area of the vessel, choking the two-phase flow and spanning the full venting of the vessel over a time longer than the 40 ms visible on the plot.

These results show that during a BLEVE, boiling may be sufficiently fast to keep pressure in the vessel during release, sustaining the release for a longer time that without liquid boiling. This explains why BLEVE should be expected to exert a significant force and impulse on a bridge. However no measurements of this hazards were made on cylindrical vessel bursting into a BLEVE. This lack of measurements and quantification of the hazard leads to a lack of modeling and prediction for hazard assessment and prevention.

This paper aims to fill the first gap mentioned above. It presents the results of ground loading measurements on a small scale BLEVE experimental apparatus, and identify some correlation behaviors between the entry parameters of the experiment and the ground force variables, with a qualitative analysis of the results and case comparison.

Experimental setup and variables

To study these details, a small scale apparatus was designed to record detailed images of the failure process and to measure overpressures very near to the vessel (within R/D vessel = 0.175/0.050 = 3.5).

The apparatus involved an aluminum tube with an external diameter D = 50.8 mm, length L =300 mm and nominal wall thickness of 1.6 mm. The apparatus and failure was designed to be representative of a cylinder failing and suffering a BLEVE at its top and centre. Fig. 3 shows the tube after failure. It is representative of the failure of an industrial size horizontal cylinder BLEVE (see Fig. 1)

The apparatus was designed and constructed to study the early moments of a BLEVE in a weakened horizontal cylinder. The key design objectives were: i) the failure mode should be similar to what is seen in practice with horizontal cylindrical pressure vessels ii) allow for variable L/D and weakened length L c /D iii) allow for variable failure pressure P f iv) allow for variable fill level f v) make the vessel as large as possible but small enough to be economically affordable to do many tests This apparatus consisted of the following parts:

i) aluminum tube (6061 T6 annealed to T0) with D = 50.8 mm and L =300 mm with wall thickness 1.6 mm ii) top of the tube machined to reduce the wall thickness over length Lc to give desired failure pressure in the range 10-35 bar. iii) Swagelok end caps. One machined to accept fill and vent lines, two sheathed thermocouples (type K 1 mm diameter), pressure transducers (TC-Direct 716-072) and a high speed piezoelectric pressure transducer (PCB M101A02). The other end cap machined to have a 30 mm diameter window iv) electric heater machined to cover bottom 30 % of tube surface v) cradle to hold tube in position vi) blast plate below tube supported on high speed piezoelectric load cells (PCB M202B) vii) pencil type blast piezoelectric gauges (PCB 137A23) at various positions viii) high speed video viewing window end, tube side and top (high speed cameras available: Phantom V711, V2512, VEO710 and Photron SA3) ix) High speed shadowgraph viewing side and end (same cameras) x) electric valves to allow for purging, fill and venting the tube Fig. 4 shows a schematic of the apparatus. Further details of the apparatus can be found in [START_REF] Eyssette | Characterization and Modeling of Near-field BLEVE Overpressure and Ground Loading Hazards[END_REF].

This work focuses on the load cells capturing the ground force signal. The load cells are screwed and pre-constrained under the base plate on which the tube rests. The sensors are piezoelectric load cells placed in each four corners of the based plate holding the tube in place (Fig. 5). The ground force is studied together with the transient pressure inside the vessel, measured by a piezoelectric transducer mounted on the filling end of the tube. It has to be noted that only the compression force was measured by the load cells; any negative effect measurement is not reliable since the mechanical properties of the screws are unknown.

The sensors characteristics are given Table 1:

Results

In the scope of this paper, 22 tests led to relevant ground force measurements. The parameters varied though this experiment are the failure pressure P fail (from 11.7 bar to 32.7 bar), the amount of liquid inside the vessel, or liquid fill level ˚liq , in volume fraction of liquid (from 0 to 87 %), and the weakened length through machining at the top of the tube, or cut length L c (from 50 mm to 150 mm). The experimental matrix is given Table 2 From our current knowledge of BLEVE, the ground loading considered in this work was due to the pressure and momentum forces caused by the opening of the top of the vessel and the release of the contents. The release was very rapid and explosive. The release produced shock waves. As the vessel opened at the top the vapour space was ejected with explosively expanding vapour and this was followed by the powerfully flashing liquid and the expanding 2phase mixture.

As the vessel failed a jet of vapour was released as described in [START_REF] Birk | Analysis of BLEVE overpressure using spherical shock theory[END_REF]. This jet expanded and went sonic just beyond the edge of the failure opening. The jet continued to expand and go supersonic beyond the opening. This jet pushed on the atmosphere and at some point it pushed strongly enough to produce a shock wave that traveled into the surroundings at supersonic speed. The loss of vapour from the vapour space caused a pressure drop that caused the liquid to begin to flash at the liquid surface. This flashing wave grew downwards into the liquid. This boiling wave emptied the vessel. Previous studies [START_REF] Baker | Explosion Hazards and Evaluation[END_REF] suggest the flashing liquid process does not produce shocks waves. However, this does not mean the liquid boiling is harmless in the explosion. It is understood that the dynamic loading resulting from the boiling is very significant to propel vessel fragment. Similarly, we assume that it has a major role in generating a destructive ground force. This hypothesis will be commented under the light of the results in this experiment.

Frequency response of the system with regard to load signal measured

A first validation of the results was made by comparing the signal obtained with the transfer function of the simplified equivalent spring-mass system of the plate mounted on the four load sen- sors (Fig. 6). The tube is mounted on a plate weighting M plate =6.3 kg. With a stiffness per sensor of k sensor = 2.8 kN/m, the natural frequency of the system is 6.7 kHz.

f 0 = 1 2 4k sensor M plate = 6.7kH (1)
The system behaves like a low-pass filter with this cut-off frequency. This value was compared to the Fourier spectrum of a ground force measurement signal (Fig. 7). All the significant contributions to the ground force act in a frequency range way below the natural frequency of the system (<1 kHz for most of it, up to 2 kHz for smaller contributions).

Air burst and low liquid volume content burst (<25 %)

The discussion of the liquid and vapour contribution to the ground loading starts with the study of an air burst with the small scale apparatus, to get a sense of what a vapour ground force may be. The ground force signal of this case is presented together with the internal pressure measured at one end of the vessel (Fig. 8a). The tube after failure is shown Fig. 8 right. Time t = 0 corresponds to the start of the crack opening of the tube. The pressure starts dropping after 0.5 ms at the sensor, which corresponds to the time for the expansion to propagate from the tube top center, where the crack opens, to the sensor location (0.43 ms). Pressure then drops below atmospheric pressure, before increasing again to this stable value (t =4 ms). The ground force peak is 9.6 kN. It presents a first peak at 3 kN, followed by the main peak. Its duration is 3.3 ms. The maximum ground force obtained for this vapour burst at 13 bar is approximately 10 kN. It is observed 1 ms after the internal pressure dropped to its minimum. An interpretation of this delay is done by expressing ground force as F = Internal pressure x Horizontally projected area of application of this pressure. Initially, the pressure is high when a small projected area becomes available at tube opening. The force increases consequently. Then, the pressure drops, but the opening area increases fast enough, such that ground force keep increasing up to its maximum. Finally, pressure is back to ambient conditions, surface area stabilizes, force drops. However, the behavior of internal pressure being taken in one point only limits the conclusions on that point.

The tube after rupture shows that the compressed air burst did not flatten the vessel as it is seen commonly for BLEVE, unlike all cases with liquid propane present in the tube. It illustrates one destructive characteristics of the liquid boiling at that scale.

Another interesting case in this study was made with a low liquid volume fill of propane (5% of liquid). The order of magnitude of the maximum peak force is the same (9.5 kN), even though the failure pressure is higher for the second case (Fig. 9a). The internal pressure profile shows a steady drop down to atmospheric pressure. The tube was more flattened than with the air case, which means there was more energy in the vessel to open it up (Fig. 9a). This may be due to the 5% of liquid boiling. Moreover, a second peak occurs on the ground force signal after the main peak, reaching 6.3 kN. This may illustrate the liquid boiling process, with a smaller peak because of the small quantity of liquid.

Similar behaviour is observed on five other cases in the range of 15-20% liquid volume fill. The contribution of each phase can be observed on one test with graphical approximations (Fig. 10). In the following figures, the liquid and vapor contribution are used only for illustrative purpose to help understanding the argument. These are graphical approximations, they do not account for a complete and accurate description of the physics of the opening process. The data collected do not allow analyzing the phenomenon into such details yet.

Violent boiling profiles

For a majority of cases with larger filling content, the ground force recorded ended up being two to five times larger than the vapour dominated cases. The clear two peaks noticed in the case of low liquid content is not visible anymore, and is replaced by a single large peak (Fig. 11).

When observing the transient pressure inside the vessel, many cases reveal a pressure rise after the initial pressure drop. This pressure rise is characteristic of the superheat boiling repressurizing the vessel. The transition from pressure drop (expansion of the vapour and superheat of the liquid) to pressure rise (boiling of the superheated liquid) is clear on Fig. 11. This transition happens simultaneously with the start of the main ground force increase. The correlation between the two events leads us to believe that the liquid boiling process contributes strongly to the main ground force.

Another feature noticed with several cases is that the maximum peak of the ground force is synchronized with the time at which the vessel ends up fully flattened. On the images of Fig. 12, we see the vessel from the radial view, at different times, starting right at the opening (t = 0) until the tube is fully flattened (t = 2-2.5 ms). This time range corresponds well to the time at which ground load signal reaches its maximum. A physical interpretation of this correlation is the following: the pressurized flow generates a vertical downward force on the tube that becomes maximum when the area exposed to the ground is maximum.

Load associated to asymmetric opening

In some tests, the initial crack didn't lead to a symmetric opening of the vessel. Only one side of the tube was flattened while the opposite remained cylindrical (Figs. 13(b) and 14 (b)). Both tests with 50 mm cut length presented the asymmetric behavior, and only those tests. Some references state that it is the limit condition to actually generate a BLEVE with a cylindrical vessel [START_REF] Birk | Blast overpressures from medium scale BLEVE tests[END_REF]. The consequence on the vertical ground force is an uneven distribution of the load on the different load cells (Figs. 13(a) and 14 (a)) compared to other cases with a more symmetric opening (Fig. 7(a)). More importantly, the main direction of the ground force was not vertical, but tilted toward the center of the opening (30 to mounted to measure a vertical force only. This means some information is missing on these two cases for the full characterization of the ground force. Two major differences between these two cases are the liquid fill content and the behaviour of the transient pressure in the vessel: it rises after the initial undershoot for the case with more liquid Fig. 13a), and drops continuously for the case with low liquid content (Fig. 14a).

All the experiments show the same fragmentation pattern: crack opening at the top and symmetric flattening. This is the most common opening pattern for a cylindrical vessel engulfed in fire. It might also be considered as the worst case in term of ground force hazard. The only variation of pattern observed through experiments is the asymmetric opening mentioned in the previous paragraph, for two experiments with short cut length.

Presence of a small first load

On some cases, the presence of a small initial load before the main ground force peak was noticed. The cases have been sorted in categories based on the presence or absence of this initial load (Fig. 15). The initial load ranges from 0.7 kN to 3.1 kN, while the main peak of the ground force ranges from 10 to 55 kN. The small load, when present, is less than 10 % of the maximum ground force peak.

The presence of the initial small load was reported for all relevant tests based on their failure conditions, by plotting the initial conditions of all relevant tests on (fill -Pfail) plane (Fig. 16). For explosions with large cuts (150 mm) the initial load was generally present with a combination of high failure pressure and high fill level. Explosions with smaller cuts do not obey that rule, as they seem to no trigger the initial in general. Finally, two outliers in the low fill -low failure pressure side are noticed as well: a case mostly vapor (Fig. 9) and a case with 20 % liquid and 18 bar failure.

The focus is made on the latter outlier (Fig. 17). When comparing the time at which this initial load appears and disappear with high speed imaging from the side of the vessel, the load seems to start when the crack, initially propagating longitudinally, reaches the ends of the machined lengths of the tube, to then start propagating circumferentially (Fig. 17 t = 0.4 ms). The change of propagation direction in the crack may be the origin of a change in the rate of growth of the opening area. This is an important parameter to consider if the ground force is visualized as the thrust through a nozzle of increasing cross section area. The small load ends during the circumferential opening process. When comparing the small load with the expansion of the cloud, it is clearly in sync with the initial vapor exit while the main component of the load corresponds to the appearance of the thicker boiling cloud coming after. A correlation can be made between these phenomena: when the vessel starts opening, the compressed vapor goes out of the crack, generating a force through a small opening area. The crack continues to propagate, opening a larger area for the fluid to expand, while the tube progressively gets flattened horizontally. At the same time, the liquid boils and is propelled out of the vessel. This observation leads to conclude that initial ground load would be due to the first instants of vapor expansion, while the main load is from the liquid boiling.

However explaining the initial load this way does not explain the strength of the ground force observed with the air case (Fig. 8) and goes against the argument made for the vapor case (Fig. 9). The comprehension of the presence or absence of this initial load remains unclear to some extent, requiring extra tests to confirm or infirm these hypotheses.

Parametric analysis of maximum ground force and impulse

From the signals observed before, three main characteristics were extracted: maximum ground force, positive impulse of the main force contribution, and duration of this positive impulse. They are plotted against the experimental variables (failure pressure, liq-uid fill level, cut length), to gage the influence of each parameter on the measurements, to present a preliminary dimensional analysis.

Influence of failure pressure on load variables

The behavior of maximum force, impulse and duration of impulse is plotted in Fig. 18. To observe relatable results, the focus was made on tests with similar fill levels, between 50 and 70 %. The general trends observed are the following:

-Maximum ground force increases linearly with failure pressure when observing the results with similar cut length (150 mm). A few low pressure cases (14-19 bar) show similar maximum force. This can be interpreted as follow: for strong failure pressure (>20 bar), force can be seen as a pressure applied to a constant surface area. Thus, the stronger the pressure, the stronger the force. The change of behavior below 20 bar is unexplained yet. It may be related to superheat limit temperature (around 53 • C for propane, near 18 bar). -Duration of impulse decreases linearly with failure pressure for similar cut length (150 mm). The higher the pressure, the higher the temperature and the more violent the boiling will be, leading to a faster emptying of the vessel. -Impulse increases also linearly with failure pressure for 150 mm cut cases. A few low pressure cases (14-19 bar) deviate from this general trend, with impulse varying against failure pressure. Impulse being the integral of force over time, the stationary behavior of maximum force at low failure pressure combined with constant linear relation between time and failure pressure lead to this change in behavior.

Influence of liquid fill level on load variables

The behavior of maximum force, impulse and duration of impulse is plotted in Fig. 19. To observe relatable results, the focus was made on tests with similar rupture pressure, between 17 and 19 bar. The general trends observed are the following:

-Maximum ground force increases with liquid fill level. More liquid means more matter expanding to build up the pressure that is applied to the vessel wall during and after the flattening process. -Duration of positive impulse is constant or slowly increasing with increasing liquid fill level. This is counterintuitive at first, as once would expect that more liquid takes longer to be evacuated from the vessel for similar opening. There is no explanation to this behavior yet. -Impulse, as expected is a combination of the two previous behaviors. Thus it increases with liquid fill level, as the maximum ground force gets stronger.

Influence of weakened length on load variables

Four cases were compared because they have similar failure pressure (around 19 bar) and fill level (around 60 %), but different cut lengths. The behavior of the force measurements with respect to the weakened length is plotted in Fig. 20. Maximum force and impulse increase together with weakened length, while duration of positive impulse decreases. An explanation for this could be that larger the weakened length, the larger the surface area on which the boiling liquid can exert its pressure, increasing the maximum force. On the other hand, the larger the area of release, the quicker the vessel empties, decreasing the duration of positive impulse. The impulse being a combination of force and duration, its behavior is dictated by the dominant behavior of these two: seemingly maximum force. It should also be remembered when observing the results for the case with 50 mm cut length that the opening was asymmetric. The fragmentation pattern may be a contributing factor to the low ground force.

Finally, a limit to this trend appears between L c =100 mm and 150 mm, where all these variables fall of the trends mentioned above. An attempt to explain this behavior could be the following: at a given pressure and fill level, for the force to be maximum, the opening should be large enough so that the surface of application of the pressure on the ground is large, but small enough to keep a large pressure inside the vessel for a longer duration due to the repressurization mechanism. However, having only four measurement points is not enough to validate this hypothesis yet. More data needs to be collected from experiments.

Conclusion

This work presented the experimental measurements of the ground force generated by a small scale BLEVE (0.6 L vessel). The maximum ground force ranged from 10 kN to 54 kN, and the impulse from 16 to 60 N.s. The shape of the signals synched with the transient pressure in the vessel showed the potential contribution of the vapour expansion through a partial opening and the liquid boiling on the fully opened vessel.

The contribution of liquid and vapor phase is not clear yet. No general behavior could be identified within this set of data. When looking at a case with air only or mostly propane vapor, the ground force generated is similar to some cases with more liquid. However the strongest loads are observed with large liquid fill content and strong failure pressure. Small initial loads observed in some cases coincide with vapor expansion, confirming that liquid boiling may be the main contribution of the ground.

Finally the interpretation of the behaviors of load variables (maximum ground force, duration of impulse and impulse) with the experimental parameters (failure pressure, liquid fill level and weakened length) show that failure maximum ground force are positively and linearly correlated to failure pressure and liquid fill level.

Simple modeling methods needs to be implemented with dimensional analysis in order to make these results useful to industrial size vessels. Then larger scale experiments are required to provide extra data for modeling, as well as validating the behaviors observed with small scale experiments. The ground force is expected to scale up with the dimensions of the vessel mostly. Thermal stratification was not observed in small scale. It is not expected to change the behavior of liquid contribution, and if it does, it should probably reduce it just slightly. But these statements require validation. The end purpose of this work is to predict the ground force generated by the BLEVE of road tankers similar to the case of Bologna 2018, in order to find technical solutions to prevent the damages from it, or implement the proper regulations concerning this hazard in the transportation and storage of pressure vessels.

Fig. 1 .

 1 Fig. 1. Opened vessel and collapsed bridge after a tanker truck accident leading to a BLEVE on an elevated highway near Bologna, August 2018. Picture from the website of Italian firefighters[START_REF] Vigilfuoco | BOLOGNA (BO) Loc. Borgo Panigale -Esplosione autocisterna[END_REF].

Fig. 3 .

 3 Fig. 3. Image of opened tube with flattened part still attached to ends. Failure runs along top of tube and then turns circumferentially at the ends of the weakened length.

Fig. 4 .

 4 Fig. 4. Schematic diagram of tube apparatus (tube D = 50 mm).

Fig. 5 .

 5 Fig. 5. Load cell mounting under based plate (left) and load cells location around the test tube seen from above (right).

Fig. 6 .

 6 Fig. 6. Equivalent mass spring system.

Fig. 7 .

 7 Fig. 7. a) Total load as the sum of the 4 load signals (P fail = 26.9bar, ˚liq = 61%, Lc = 150mm); b) Fourier transform of the load signal, compared with the transfer function of the critically damped equivalent mass-spring system.

Fig. 8 .

 8 Fig. 8. Air case rupture (P fail = 13bar, Lc = 150mm, filled with compressed air) a) Ground force and internal pressure measurements after failure; b) Shape of the tube after failure.

Fig. 9 .

 9 Fig. 9. Propane vapour rupture (P fail = 16bar, ˚liq = 5%, Lc = 150mm) a) Ground force and internal pressure measurements after failure. Dashed lines show the measurements for the air case, for comparison purpose; b) Shape of the tube after failure.

Fig. 10 .

 10 Fig. 10. Internal pressure and ground force from rupture of low liquid content vessel (P fail = 17.7bar, ˚liq = 17%, Lc = 150mm) a) Ground force and internal pressure measurements after failure; b) Shape of the tube after failure.

Fig. 11 .

 11 Fig. 11. Internal pressure and ground force from ruptures of high liquid content vessels (a -b): P fail = 30.6bar, ˚liq = 64%, Lc = 150mm; (c -d): P fail = 26.9bar, ˚liq = 61%, Lc = 150mm) a-c) Ground force and internal pressure measurements after failure; b-d) Shape of the tubes after failure.

Fig. 12 .

 12 Fig. 12. Comparison of events on the ground load signal with the high speed imaging radial view at various times (P fail = 18.6 bar, ˚liq = 57%, Lc = 75mm).

Fig. 13 .

 13 Fig. 13. Case with small weakened length and side opening (P fail = 19bar, ˚liq = 56%, Lc = 50mm): (a) Measured ground force and internal pressure signals; (b) tube after failure.

Fig. 14 .

 14 Fig. 14. Case with small weakened length and side opening (P fail = 19.1bar, ˚liq = 16%, Lc = 50mm): (a) Measured ground force and internal pressure signals; (b) tube after failure.

Fig. 15 .

 15 Fig. 15. Ground loading measurement, gage by gage and total sum for 3 different profiles: a) No small load: P fail = 17bar, ˚liq = 60%, Lc = 150mm. b) Progressive load build up: P fail = 18.6bar, ˚liq = 56%, Lc = 75mm. c) Presence of a first small load: P fail = 26.9bar, ˚liq = 61%, Lc = 150mm.

Fig. 16 .

 16 Fig. 16. Test summary with specification of the presence or not of initial load, symbols differentiate cut lengths (a) No initial load; b) Initial load merged with main load; c) clear separated initial load).

Fig. 17 .

 17 Fig. 17. Pressure and load signals synchronized with radial view of the vessel, frames corresponding to beginning and end of the initial small load (P fail = 18.2bar, ˚liq = 20%, Lc = 150mm.

Fig. 18 .Fig. 19 .

 1819 Fig. 18. Behavior of (a) maximum load; (b) duration of positive impulse; (c) positive impulse, with respect to failure pressure, for cases with liquid fill level between 50 % and 70 %.

Fig. 20 .

 20 Fig. 20. Behavior of (a) maximum load; (b) duration of positive impulse; (c) positive impulse, with respect to weakened length, for cases with failure pressure between 17 and 19 bar.

Table 1

 1 Ground load sensor characteristics.

	Sensor use	Designation	Range	Response time	Uncertainty
	Ground loading	PCB M202B	0-44.5 kN	60kHz max frequency	< 0.2 % Full scale (0.01 kN)
	Transient internal pressure	M101A02	0 -34.5 bar	1.5 s

< 0.4 % Full scale (138 mbar)

Table 2

 2 Experimental Matrix.

	Test #	Cut Length (mm)	Pfail (bar)	vol fill (%)	Test #	Cut Length (mm)	Pfail (bar)	Fill (%)
	2	150	11.7	52	15	150	15.5	air
	3	150	13.4	55	16	150	16.0	5
	4	150	15.6	59	18	150	18.5	61
	5	150	16.7	63	19	150	30.6	65
	6	150	17.0	59	20	150	26.9	63
	7	100	18.8	58	21	150	19.3	86
	8	100	12.5	57	22	150	23.3	87
	9	50	19.0	55	23	150	32.7	70
	10	50	19.1	15	24	150	26.3	24
	11	150	17.7	17	25	150	29.5	18
	14	150	18.3	18	26	75	18.6	57

Acknowledgments

This work was a collaboration between the Mechanical and Material Engineering of Queen's University in Canada and the LGEI at IMT Mines Alès in France. It was possible thanks to the funding from the Natural Science and Engineering Research Council of Canada with a discovery grant, and the help of the technical team in labs of the Institute of Risk Sciences at IMT Mines Alès, namely Pierre Lauret, Laurent Aprin, Christian Lopez and Pierre Slangen. Dr Stephane Corn and Arnaud Regazzi are also acknowledged for their help in the frequency analysis of the experimental apparatus.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.