 We can start the webinar today. I will give immediately the floor to our director. As usual, Alexis Gousdel for the opening remarks. Alexis, the floor is yours. Thank you. Thank you, Marika. Good morning, everybody. It's a real pleasure again to see so many people joining us from everywhere in Europe and beyond Europe for a new edition of the EMCDD webinars. I would like also to thank our guests who are joining us for this edition of the webinar today. Frédéric, Tim and Sarah. And also Jean-Mathias, who is going to be the chair of the webinar today. I think this webinar comes at a very important and very interesting moment for many reasons. The first one is since the Covid pandemic preparedness at the European level has become even more central in most every areas of policies of the EU, starting, of course, with public health, with I think the European Union, meaning the institutions, but also the member states, to some extent rediscovering the importance of public health, which after years and years of cuts in the budget at national level, have shown that at some stage, the public service, the well-being that it is supposed to guarantee to our citizens was at stake, and that basically, even if we are considered at global level as a group of rich countries, or at least richer countries than others, we were almost paralyzed in our capacity to address such an important event like the Covid pandemic. And it has been a big wake-up call for many institutions, including at national level. But it's much more beyond Covid, just to take another example, there is the war in Ukraine following the invasion of Ukraine by Russia. We see that the impact is also global, not only in terms of peace, in terms of safety, but in terms of migrations, in terms of impact on food for the entire planet, especially for the poor countries on the planet that are extremely depending to a big extent from the production of cereals from Ukraine and from Russia. So this shows that the world is even smaller than before. We just announced a few days ago at UN level that we have passed eight billions of inhabitants on this small planet. And at the same time, we see that there is a need for a state of preparedness for managing the migrations or defining a framework that allows to treat everybody in line with the European values and the fundamental rights, but also public health. But today preparedness is also about the economic crisis or the energy crisis. So it's a term that has invaded all the policy areas, not only at European level, but also internationally. So that's the first thing. For EMCDDA, it's a very important word already, and it will become even more important in the future, because as you probably know, the European Commission early this year has made the proposal to change the mission, the mandate of the agency to transform it in a full-fledged European drugs agency with a broader mandate. And one of the chapters in the legislative proposal is proposing new competences targeting or contributing directly to preparedness. But I would say overall, sorry, the main change, the future change with the new mandate of the agency is that preparedness of the EU as far as drugs use consequences and so on are concerned will be the new mission, the new goal of the agency. And this means that we will need to design over the coming years the new services that the agency will provide for the EU, the member states and the professionals and the citizens. And part of that has a direct link with wastewater in many ways. First of all, because wastewater today has been clearly identified and that's the topic for the webinar today as a key element for preparedness, for ensuring preparedness. And we also have discovered that wastewater analysis could be used also to detect the COVID virus in some countries or in some cities. It's now mushrooming the opportunities and the recognition of what wastewater analysis epidemiology technology can bring. I still need to tell you that when we first heard about wastewater analysis, it was 21 or 22 years ago here at EMCDDA. It was like kind of a fuzzy idea of a mad scientist, a bit like, as I am Belgian, you probably know, we are famous for Belgian comics and Tintin. And in the books of Tintin, there is a crazy professor who is inventing mad things. And for us, the first time we heard what? Using the water from the toilet to do epidemiology. Okay, shall we call the psychiatrist or the emergency room? And today, that's a scientific discipline, well established. And we are very proud at EMCDDA that we work with all the colleagues who are involved and certainly as part of the new mandate of the agency. We are considering, we are planning, we start drafting the plans for future implementation. There are two elements that we support, I hope, and I'm sure in the future, the work on wastewater and the cooperation with EMCDDA. It's the possibility and the means for the agency to develop better and better support, complement the resources of data collection and wastewater is the first of them, not the only one, so we need to see how we can achieve that. But also, it will be the definition, the creation, the establishment of a European network of forensic toxicology and pharmacology laboratories that will need to link the work, new areas of work, new study, new research, new data collection, but also to link with wastewater, to link with the work that is done on drug checking, for instance, the work that is being done on the residues of syringes, many other fascinating projects that for the moment we are just able to help with small budget limited means and we will have that opportunity to explore together how we can move forward, how we can together build and contribute to a better preparedness, because definitely drugs are everywhere. Everywhere can be used as a drug. Everyone can be personally or indirectly facing problems of addiction at some time in their life. It's time to realize that drugs, when we talk about drugs, it's not only about heroin injection. And therefore, if we want to be prepared together, I think we have a wide range of opportunities as far as, for instance, is concerned the cooperation on wastewater epidemiology and analysis. So for that reason, for those reasons, I'm very happy and pleased and supportive for Marika, Jean, all the colleagues and our guests and for all of you who are joining us for this meeting today, because that's not the most famous method for collecting data yet, but certainly this is already one of the pillars of what allows the MCDDA today and even more in the future to speak about not only how was the drug situation in the EU three, four, five years ago, but to speak about what is happening about substance use in some places or some cities in Europe, almost in real time. I know it's not on permanent basis, but that's the part that allows us to know what's happening now. And therefore, we need to strengthen this pillar and to further develop. And I'm sure that the three speakers today will share with us their ideas and help us to understand and a bit anticipate how could look like this cooperation in the future. So I would not be able to draw the conclusions. Honestly, I don't think you need me to draw the conclusions. You are the experts. So I'm looking forward to the discussion. I will listen to your presentations. Then I will need to escape. So I will not say bye-bye. I want to thank you again and full success. And I look forward to the meetings maybe next week at the occasion of Lisbon Addiction. I hope we can meet some of you at the conference. Thank you very much. Thank you very much to you, Alexis, for your always illuminating words and for your time in such a busy period. And I give the floor directly to João Maties to start the webinar. Thank you very much. Thanks, Marika. And thanks, Alexis, for the introductory words on this webinar. Hello to everybody and welcome. I see now that the chat is working. Thanks, Marco, for starting that out. And I can see that we have people from all over. So it's really great. I think it's one of the, let's say, the meetings we have here at TMCDA that we have a broader audience from all the continents. And I think it's quite valuable for us. First of all, I would like to thanks also Marika, Alessandra and Marco for the help organizing this webinar for me. It's super easy. It's my third one. It's super easy because they make my life so much easier. And I really need to point this. It's a pleasure to chair a session on wastewater. I've been working quite closely with the SCORE Network for quite some time here internally. We have been working for more than a decade with researchers all over Europe, but also from other parts of the world, Australia, US, developing the methods. That has established itself not only as a complementary method, but as an indicator on its own, providing, as Alexis said, almost real-time data on the recent trends on drug use, which for us, it's key. And we believe it's important to have this type of information to better inform policy and practice. I'm very happy to have the three speakers today that we have here because we try to have a different focus from the traditional substances covered from wastewater-based epidemiology. And we would like to have three different presentations focusing on how wastewater can inform us on substances that are traditionally not covered in our traditional epidemiological tool. So I think it will be quite interesting to have and listen to these presentations. We will have the first speaker, Frederick Bin, which is an assistant professor at the University of Amsterdam and also a researcher in KWR Water Research Institutes from the Netherlands. Then we will have Tim Bogertz from Belgium, PhD researcher in the Toxicological Center from the University of Antwerp. And last but not least, Sara Castiglione, head of the Environmental Epidemiological Indicators Laboratory in the Mario Negri Institute in Italy. And Sara is also the coordinator of the SCORE Network, which is the wastewater network of researchers all over the world. We will move to the first speaker. We will cover the question on how wastewater contributed to the emergency response to COVID-19 and also how in the drugs field, it can contribute to monitor new potential threats with a focus on crack cocaine. So I will give the floor now to Fred, so please take your time. And after the three presentations, we will have space for questions. So you can use the Q&A button at the bottom of your screen and write down your questions. And at the end of the three presentations, we will give the space for the speakers to address the questions you might have. So Fred, the floor is yours. Thank you very much, Joao. I hope you can hear me. Yes, excellent. So thank you very much, Joao, for the introduction and also many, many thanks to Alexis for the great introduction and actually also the trust that he and EMCDDA as a whole has put in this crazy idea that someone had 20 years ago about trying to use wastewater for public health purposes. And yeah, as Joao said, my presentation actually will cover two very different topics, but the connection between the two is obviously wastewater. So on the one side, I will give you a quick overview of what has been done with wastewater when it comes to monitoring the virus that caused the COVID-19 pandemic. And then I will switch completely and come more into the topic that you're used to with respect to also the work that the EMCDDA does. And specifically, I'm going to talk about crack cocaine. But first of all, while you might already have understood it from both Joao's and Alexis' introduction, why is wastewater so interesting? It's not just about us crazy scientists in the laboratory trying to come up with new ideas of things to do. But it's because there is actually a real interest and wastewater is a huge source of information about the lifestyle and the health status of large populations. And the reason for that is that everything that we do in our lives, all the substances that we come into contact, but also the pathogens or the diseases, the agents of disease that we come in contact with either voluntarily or involuntarily, they will leave traces in our body. And in particular, they might leave traces in our urine or our feces. And obviously, by going to the toilet and flushing the toilet, all these evidence, these traces of the things that we come in contact with will come into a sewer network and the sewer network will convey all of these waste, wastewater to a wastewater treatment plant. And at that stage, we can collect samples together, obviously with the wastewater treatment plant personnel that provide us these samples. And in these samples, we can analyze a number of things, biological things, pathogens, viruses, microbes, but also chemicals like drugs or environmental contaminants. And this is basically the principle of wastewater. And I just mentioned pathogens, chemicals, as you can see from this map, very overwhelming, I apologize, but it gives you a quick picture of all the things that you can that we can already or that we could potentially measure in wastewater. And as you can see on the left, it covers all the part of illicit drugs. Then you have pharmaceuticals and so-called personal care products. So everything that is being used in our everyday activities in consumer goods that we have in pharmaceuticals. And then you have other markers that are related to the population, industrial chemicals like pesticides and things like that. And then also markers that are related to our overall well-being, like stress markers or food markers that indicate what kind of diet a person follows. And then finally, biologicals. So everything that is related to viruses and bacteria, as I mentioned before. So as you can see, it's really broad and there is a lot of information that can be gathered from analyzing wastewater. So then let me get into the first application that I want to talk about. And that's something that probably a lot of you have heard about because actually, despite the fact that wastewater-based epidemiology or the use of wastewater for tracking human health was around for a while, when the SARS-CoV-2 and the COVID-19 pandemic arrived, it actually gave a huge boost to the use of this approach to monitor public health. But we actually had been doing this for a while already with illicit drugs together with the EMCDA, but also in other fields related to environmental chemicals. So as you all remember, there was a large issue with COVID in particular with SARS-CoV-2 related to the fact that people were walking around carrying the virus but not showing any symptoms or they were only showing very mild symptoms and also the fact that people would be infectious 48 to 72 hours before they actually started developing symptoms. So this meant that the virus might be transmitted to people without them knowing, without them having any symptoms. And this made it much more complicated to monitor the current situation to prevent the disease from spreading. And so that's where some researchers and colleagues, actually colleagues of mine here in the Netherlands came up with the idea of trying to track the virus in wastewater. And the principle is very similar to what I just mentioned before. So the fact that people are affected with the virus will leave some traces, some evidence of the virus in their body, in this case in particular in the feces. And obviously again, by going to toilets and by collecting samples at a wastewater treatment plant, it is possible to find traces of the virus in the wastewater. And by doing that on a large scale on a big wastewater treatment plant and doing that over time, it is possible to actually say something about trends in the population, whether the virus is going up or the virus is going down in the population of a city or that a population that is served by a wastewater treatment plant. And well, this is how it works. And this applies to the wastewater to the virus but also to drugs, as I mentioned. So as you can see here, a very schematic overview. We have sewer networks and sewer networks serve domestic users. So toilets but also showers, dishwashers and all of the things. And then we also have industries and other activities, other human activities that are also generating wastewater that is being fed to the wastewater treatment as to the sewer network. So what we do is that before the wastewater treatment plant, just before the wastewater is actually treated, we collect samples over a course of over a certain period of time. Most of the time is done over 24 hours because we want to have a picture of what happened during that specific day. So on the right side, you can see a picture a bit small maybe but that's what we call an auto sampler. So it's a device that allows us to collect on a regular basis, samples directly from the wastewater stream before the wastewater is treated. And then we can collect these samples and analyze them in the laboratory. And this is what you can see here. So what has happened is that the samples are being collected, they're brought to the laboratory and they're being pre-processed. Here we centrifuge them and then we process them further to extract the genetic material of the virus. We do an amplification with this system, with this PCR, which is the same system that was used to do conventional testing on people to see whether you were infected with the virus. And then we, here you have a colleague of mine who's at the KWR who's analyzing the outputs. So the results of the data to see if he could find traces of the virus in wastewater. And what was done later in the Netherlands, this is the example of the Netherlands. So the Institute of Public Health and the environment of the Netherlands, together with the Dutch government and the wastewater treatment plants set up a huge sampling campaign which is still ongoing today where they collect wastewater samples of each of the wastewater treatment plants in the Netherlands. And I'm talking about roughly 300 wastewater treatment plants in the whole country. And they collect daily samples and they measure constantly the amount of virus that is present. And they use it as an indicator, as you can see here, to give an idea about the amount of virus that is circulating in the population. And currently in the Netherlands, testing of individuals is done only for specific categories of people. For example, if you work in hospitals or you might come in contact with vulnerable people. But for the rest, for the common citizen, there is no more government generalized testing. So wastewater is used actually as one of the very few indicators currently to monitor what is going on in the population. And as you can see on the right side of this graph, this is really the data from just a few days ago showing how the situation in terms of the amount of virus at a country level is currently. And then I'm gonna move on to the next part of my talk and that's about crack cocaine. So completely different still related to wastewater obviously but a completely different topic. And here again, the principle is the same. So we collect samples at the wastewater treatment plant. In this particular case, we focus on urine because cocaine and crack cocaine are mainly leaving traces in the human body in urine, not in feces like the virus. And the difference with the virus is with what I presented just before is that once we have measured the amount of the drugs or the drug residues in wastewater, we actually relate this to the amount of people that live in a certain city. Actually, I forgot to mention, we do this for the virus as well. And the reason for that is that if you're monitoring two cities that have different sizes in terms of population, obviously the total amount of the substance or the virus might differ but this is not due to increased use or increased infection. It's just due to the fact that you have a larger or a smaller population. So we divide basically the amount of the substance or whatever we measure by the population and then we use this to do comparison between cities. And the reason why I mentioned this is because in a second, I will show you some graphs where we are comparing cities between each other which have different sizes. So crack cocaine probably known to some of you or not everybody, but it's a form of cocaine that actually can be smoked. And because it can be smoked, it causes, it has some additional risk, health risks and with respect to addiction compared to conventional cocaine consumption by sniffing. And there was an interest about monitoring crack cocaine also with respect to the work that we do together with the EMCDDA because there were indications that there were an increased number of people requiring treatment because of crack cocaine. But at the same time, it was being difficult to determine whether this was really the case and to get us some more information about where this might be happening and if it might be happening. So is there really an increase in the use of crack cocaine and the fact that there was no clear picture about it. So here is where wastewater could come into play and give some additional information on whether we can find some overall community wide information about the consumption of crack cocaine. So this is what we did in this study. So we collected wastewater samples from different cities and we looked into certain chemicals that are formed when you smoke cocaine. So I don't want to go into the details of the chemistry but just as you can see here in the middle you have a figure of the structure of the molecule of cocaine and what actually happens when you heat it and that's what happens when you're smoking it you transform cocaine and you form so-called biomarkers or transformation products. So you slightly modify the molecule of cocaine and you create new molecules and these molecules are very specific to the smoking and they are also being released in urine and we can trace them in wastewater. So this is what we did. And here I just showed you a glimpse of some of the results that were achieved by collecting samples across different cities in Europe. I hope you can read them and it's not too small but we had samples from the Netherlands, from Belgium, different cities in Spain and in Italy and also in Dublin. And what we actually realized, first thing that we actually saw is that we could find these markers in basically all the cities that we monitored. Also, there was some indication that some cities might be more at risk or had reported more use of crack cocaine while others had no information at all about it. Well, by analyzing wastewater, the first thing that we could see is that, well, we seem to be finding evidence of crack cocaine consumption in all of the cities that we studied. And then the other thing, as you might see, the cities of Amsterdam and Antwerp in Belgium actually showed pretty high levels of those biomarkers compared to the other cities. As I mentioned in the beginning, we did take into account the fact that these cities do not have the same size in population, so these are normalized by the population. And it's interesting to know obviously that, well, the fact that we find increased crack cocaine consumption in Antwerp and Amsterdam might also be related to the fact that these two cities are also among those that show the overall highest consumption of cocaine. So maybe there is also just a link between the availability of cocaine in general and the consumption of crack cocaine. But so interesting was actually really to find traces of this everywhere we looked for it. And then using data, well, using actually samples from Amsterdam that we have collected over the course of the years, we also wanted to look whether we could see increasing trends because there seemed to be an indication that consumption might be going up. Although in this study, we did not have data, historic data from other cities, we only had this for Amsterdam. But from what you can see here, basically the fluctuations, there doesn't seem to be an increasing trend. So the line in the middle, sorry, I maybe I forgot to mention, this is the median or the average, in this case is the median concentration. So the average amount that we measure over time. And as you can see, it does fluctuate a little bit, but there doesn't seem to be a clear indication that it's increasing or decreasing. And that brings me to the end of my presentation. And I would actually, before I give the word to the draw again, thank two of my colleagues. Well, one is Ruth who did a lot of the work on the wastewater analysis of crack cocaine and my colleague, who was coordinating the work on the SARS-CoV-2 virus. And thank you all for listening. Thanks so much Fred for the presentation. I think it's worth mentioning that the work done on crack and wastewater was part of a bigger European project called the UCME, as you can see here still on the slides. That's covered quite a lot of different areas in wastewater, also improving the methods to establish wastewater as also a tool for cannabis use. So it's quite broad and for me personally, because crack use, we are having signals from other indicators that there might be an increase of use in Europe. Wastewater I think will be, or was the first that actually gave us some quantitative information about this. So I'm super grateful for the work that came out of this project. Thanks Fred. We will move now to the second question that will be answered by Tim. And the question is, what was the impact of COVID-19 on drug use with a specific focus on monitoring opioids in Europe? Which I think also we know about the opioids epidemic in the US. So it's a question that we get quite often if wastewater also can be used to assess the use of opioids. So Tim, please, the floor is yours. Right, thank you Chvau. So to answer this question, it's a really broad answer that I have to give for this because there are discrepancies between different data sources and it's also dependent on the country and the compound itself. So we will apply wastewater analysis to monitor the consumption of different substances, including drugs and opioids. And we will try to give an answer to this question. So let me go to the presentation. Antwerp, a place where many people live their lives, a place where people not only come to work, but also to relax from a day of shopping on the map to a nice terrace in the city center. Below the sidewalk, there's a world of a completely different kind, the sewer. In this kilometer long network of pipes, we can find the remains of everything we do, consume and wash away. My name is Tim Boyars, PhD student at the University of Antwerp and I will guide you through this mysterious world. And together we will unravel the secrets of our society during the COVID-19 pandemic. It is no surprise that the COVID-19 crisis dramatically changed our lives. Before the pandemic life was full of social gatherings, concerts, parties, and this suddenly stopped due to the restrictive interventions such as social distancing, nightlife closures, and remote work. And with the absences of all these things, it felt like there was only one thing that we could do which was going outside and taking a walk. Otherwise we remained stationed at home. This of course raises the question how the use of illegal drugs and other substances was affected by these social measures during the COVID-19 crisis. And the extent to which the use of illegal drugs and also pharmaceuticals was affected by these interventions needs to be further explored at the population level and therefore there's a need for more complementary information. Waste water can be regarded as a reflection of our society. Everything we consume and wash away ends up in the sewer system. And with everything I really mean everything from illegal drugs and alcohol to pharmaceuticals and SARS-CoV-2. From this point, waste water contains a wealth of information about our society and the consumption of different compounds. And multiple samples can be analyzed to obtain epidemiological information about different substances such as pharmaceuticals and illegal drugs in different locations and at different time points. So the aim of this study was to monitor temporal changes in the consumption of substances and pharmaceuticals during the COVID-19 pandemic. We will compare consumption during the crisis with the consumption in the period before the start of the COVID-19 pandemic. Additionally, we also investigated the potential of wastewater-based epidemiology to evaluate the effects of certain policy changes. Fred already mentioned it, but within wastewater-based epidemiology, measured concentrations of human metabolic excretion products or so-called biomarkers are transformed into population-normalized mass loads by multiplying with the wastewater volume and dividing by the population served by the wastewater treatment plant. And these population-normalized mass loads, they can serve as a proxy for consumption of different substances. Now, during the COVID-19 crisis, the population dynamics within these different wastewater treatment plant catchment areas were heavily affected by the social measures. And therefore, there is a need for an accurate population proxy to account for daily variations in population size. For this purpose, we applied mobile phone data to estimate daily population sizes. Now, let us first zoom in on the illicit drug use in Europe. And here you can see the use of cocaine in the year 2021 in different European cities. So this is from the European Wastewater Monitoring Campaign that was already mentioned earlier by Joao. And here we analyze wastewater samples from over 100 European cities. And the bubbles show the use of cocaine in each of these locations. Larger the bubble, the more cocaine that's consumed at these specific locations. What's interesting, if you look at Belgium and the Netherlands, you can see that these countries encounter the highest level of cocaine use within the European landscape. And for Belgium in particular, the port of Antwerp and its location could potentially explain why cocaine is available to this extent in this country. In contrast to Western European countries, the use of cocaine in Eastern and Northern European countries is much lower. And if you look at the use of MDMA or ecstasy, you can see similar spatial patterns in the use of this substance, with also a high levels of MDMA in the Western European countries. And this is probably because Western Europe is a hotspot for the production of synthetic drugs, including MDMA or ecstasy. And these graphs show that wastewater-based epidemiology can be used to monitor illicit drug use at a very local level, even for compounds that are not included in official figures and circulate in illegal circles. So let us now zoom in on the situation during the COVID-19 pandemic in Belgium. And these graphs show the use of cocaine in the first wave of the COVID-19 pandemic in Brussels. The Y-axis gives the population normalised mass load expressed in milligrams per day per thousand inhabitants, and the X-axis gives the different time periods of the first wave of the COVID-19 pandemic. The colour of the bars give the different phases. In red, the pre-lockdown period in 2019. In green, the lockdown period. And in blue, the exit strategy. In purple, the relaxation period. The size of the bars gives how much cocaine was used in each of the different time periods. But before we focus on that, let's take a moment to consider the different phases of this first lockdown wave. So during the lockdown period, this period was characterised by very strict measures. So people had to remain at home and had no social contacts outside of their bubbles. The catering industry and the nightlife sector were fully closed. During the exit strategy, this gradually changed and people could meet up with four persons in total. But everything else remained the same. And then during the relaxation periods, the catering industry reopened with a curfew at 1 a.m. And people were allowed to meet with up to 10 persons per household. And what's striking if you look at the height of the different bars, you can see that the use of cocaine in 2020 during the COVID-19 crisis in Brussels did not decrease compared to the period before the start of the lockdown. And this is interesting because this might indicate that even though with the presence of all these social interventions, people still continue to use drugs. And the same is true for the other locations where we collected daily influent wastewater samples. And you can see a stable or sometimes even increasing pattern in the use of cocaine. But it did certainly not decrease. And similar temporal trends were also found for MDMA of ecstasy. You can see that the use of MDMA was stable in all locations with the exception of BOM, where a higher use of MDMA was reported during the lockdown phase. And this is of course interesting because this means that the people might have been less impacted by the limited social and recreational opportunities to consume drugs. It's possible that people still continue to use drugs at home during their home confinement. The stable temporal pattern could potentially also be explained by the limited effect of the social interventions on the availability of drugs in Belgium. Of course, this can be country specific. So now I'm only talking about the situation in Belgium. And even though with the presence of all these interventions, it could also be that people still engaged in illegal parties or participated in the so-called streaming parties behind their computers. But these results indicate that wastewater-based epidemiology as an approach can be used to give information on the lifestyle of different communities during the public health crisis. And in a similar way, the human-metabolical excretion products of pharmaceuticals are also excreted in the wastewater. And this means that wastewater-based epidemiology can also be applied to monitor the use of different pharmaceuticals, including opioids. On this figure, you can see on the y-axis, the locations that we monitored and on the x-axis, the sum of the population normalized mass loads of the opioid metabolites. What you can see is that the use of tramadol and its metabolite, odesmityl tramadol, was the highest in all locations followed by codeine. And what's also interesting is that in the majority of locations, spatial differences were found for all these prescription opioids. And this means that wastewater can be employed to highlight and prioritize locations with a higher burden of opioid use. So let's now zoom in on the situation in L'Euve. In the catchment area of the University City of L'Euve, we performed a longitudinal sampling campaign for which we collected daily-influent wastewater samples between September 2019 through April 2022. The vertical axis gives the population normalized mass loads and the horizontal axis the time. The blue line on this figure shows the trend in the use of tramadol, a prescription opioid, and the red dots represents the weekly average population normalized mass loads. The yellow bars show the first and second lockdown period in Belgium. An important to know is that during the first lockdown, people were heavily restricted by the COVID-19 measures, as I already mentioned, while during that second lockdown, only the catering industry and the nightlife sector were affected by the countermeasures. And if you look at the trend line, you can see an increase in the use of tramadol at the start of the lockdown periods. During these times, there was also a decrease in the number of students present in the catchment area, and commuting was heavily influenced by the social measures. And this is also reflected by the number of people present within the boundaries of the catchment areas served by the wastewater treatment plans. And this figure gives the population number based on mobile phone estimates over time. During that first lockdown phase, there was a 50% decrease in the number of people present in the catchment area. And this also means that the demographic features of the population of Leuven changed during the COVID-19 crisis compared to the pre-pandemic periods. And with the absence of the majority of students, the population could potentially shift to a proportionally older population that uses more pharmaceuticals, including the prescription opioids. And during the second lockdown, the influence of the COVID-19 measures on the population was smaller and per capita pharmaceutical use was less affected. And similar temporal twins were also reported for other prescription opioids as visualized on this graph. So it's important to keep these demographic changes that were the result of COVID-19 crisis in mind when interpreting temporal patterns. And this is also true for the illegal drugs. What's also interesting is that over the sampling campaign from 2019 to 2022, the use of opioids slightly increased. And this is in line with other epidemiological data sources such as pharmaceutical sales data and health interview surveys that indicate that the use of these pharmaceuticals is slightly on the rise in Europe. And this also indicates that wastewater-based epidemiology can be used as a complementary information source to follow consumption patterns at a high spatial temporal resolution and give information on the burden of opioid use in different locations. So we dived together in the wastewater and we learned that it consists of a wealth of information about our society. During the pandemic, the use of illegal drugs was less affected and people might continue to use drugs during their home confinements. And the COVID-19 pandemic dramatically changed or partially changed the demographics within the different catchment areas. So isn't it fantastic that wastewater can give so much information about our lives and about our society? So next time you walk through your city center, please remember that there is a secret world below your feet, a world that contains a lot of information about everything you do and wash away, a world that reflects our society. Thank you. Thank you, Tim. I think it was a good example of data storytelling. At some point, I thought I was back to childhood and was listening to a story, which is a good thing. So congratulations. I think it was quite interesting the way you drove us through the impact of COVID-19 on drug use. We will now move to the third question. That is also a challenge for us here internally on how we can measure NPS use, so the use of new psychoactive substances. And Sara will tell us how wastewater analysis can help us monitoring NPS use in Europe. So please, Sara, the floor is yours. Thank you. Thanks, Joao, for the introduction. I hope you are hearing me. Okay. Yes. Yeah, I will deal with a specific topic within the wastewater analysis, wastewater-based epidemiology, which is related to the NPS, new psychoactive substances, that are very relevant nowadays for public health, because they may pose a serious threat to human health. The main reason is that these substances have similar effects to the classical drugs like cocaine, heroin, cannabis, because they are synthesized from the same structures, but they can be much more active. For instance, for synthetic opioids, we have a synthetic substances that are much more active than, for instance, morphine or heroin. The idea is that people synthesizing these substances are taking the chemical structure, for instance, here of amphetamine, and they are adding some additional chemical groups to modify the substances. So the main, the bone of the substances is always the same, so the effect is similar, but the substance is a different one. So the first effect is that we have hundreds of different substances with a similar structure, but with different active effects. And we know that amphetamine is not legal, so it is forbidden to sell amphetamine in shops or on the web, but for the other substances which are synthesized, this is not true. So most of them, they are legal, and so they can be sold without any problem in the market. Of course, the legislation is working on this and the situation is changing, but this is the main issue. So we have a high number of substances that make it difficult to evaluate the real prevalence of use of these substances. Several of them, they are not controlled, so they can be sold legally. And once one of these substances becomes illegal, so restricted, protected, there is another one coming in the market which is free legal and so it can be sold everywhere. So new substances are really appearing very fast in the market and the number and also the kind of substances in the market is changing very fast. The use of these substances is not related to the general population, but mainly to sub-populations like people going to festivals or parties. They use it mostly recreational. And one more issue related to these formulations, these substances is that several times they appear in a cocktail. So they are mixed more than one and the person who is taking the substance is not aware of which kind of substance is taking. And this of course is increasing the potential toxic effects. So it is very difficult, as I said before to assess the prevalence and we also have not so much information. In this framework, in this framework, the EMCDDA in collaboration with Europol started several years ago and only established an early warning system to identify in the market all the new substances and also to evaluate the health risks. This is the figure taken of course from EMCDDA, the most updated figures. And here we have the profile of new psychoactive substances detected in the market since 2005. In the beginning, we had not so many substances but then in 2014 and 2015, there were about 100 new substances detected per year in the market. Then in the last few years, this is maybe a good news that the number decreased but is still anyway high. We are speaking of more than 800, almost 900 NPS in Europe and then you have also the different classes which you can see that the profile is also changing in the beginning of 2014-15. Synthetic cannabinoids were the most abundant class and then together with synthetic catenants. And in the last years also opioids came increased the number of new substances. So wastewater-based epidemiology, as we have already seen, is based on the measurement of excretion residues in urban wastewater and is normally used to estimate the consumption of classical drugs and it can be used also for NPS. Or at least we tried to see if it could be used. And we will have the answer during my presentation. So the application of this approach is very useful because it can give objective information and this information can be updated very fast because this approach allowed to have a result almost in real time. It is very flexible because ideally we can investigate all the cities, all the towns served by a wastewater treatment plant. And we can also decide a specific timing for sampling and for the analysis as we prefer. And we can have any qualitative information and also quantitative. So we can see which are the substances used in the population and how much of each of these substances is used. For NPS we have also some challenges related to the fact that the use of these substances is less than classical drugs. And so we are respecting low levels in wastewater. We have of course a nine number of substances and this with chemical analysis may create some problems. And for several of these compounds we don't know the really the metabolism, human metabolism. So we know that they are created mostly with urine but it is difficult to identify a proper biomarker. And for most of them actually we are analyzing not the metabolites but the parent compound. So the substance itself. We for doing this, this investigation we are working on two different approaches. The first one is a screaming approach. We can do the screaming. We are screaming hundreds of different substances. Normally we are working with about 250 compounds. And in this case we don't have a reference analytical standard for each substance. So we can say that we identify a substance but we can't confirm exactly the identity of the substance. So in this case we can do a qualitative analysis. We can say which substances are in wastewater. The second approach is the target. Approaching this case we have reference material reference standards and we can of course in this case we are working with less compounds but we can identify a substance and then quantify it as we have seen for pharmaceuticals and classical drugs in the previous presentations. We started the working in the framework of this core network on NPS several years ago. And this is the first publication. We were working at that time on only on synthetic cuttings, 17 substances and we could monitor four countries with samples collected in 2014 and 15. Here we have the first results at that time in UK we had methadone as the most used substance and it was, here we have the mass loads. We have already seen and explained very well what this means. So mass loads can be compared, data from different cities can be compared if we consider the milligram per day per 1,000 inhabitants. So here is a city in UK with methadone with quite high consumption. Then Italy, methadone was the most used but at almost 10 times less and then Spain with a different provider. At that time, we could also investigate the weekly profile of use and we confirmed the recreational use of these substances because we can see that methadone for instance here and also metcathinone in Italy was increasing, the use was increasing during the weekend. Later we had two projects called NPS, Euronet and Eusimi that were founded by the European Commission, the GOM and the first one was specifically related to the investigation of new psychoactive substances using wastewater analysis and also urine analysis and the second one was more related to the monitoring of emerging drug problems among which we investigated NPS. So I am going to show you some of the results from these two main projects. The first one, we collected raw wastewater samples from 14 European countries and we could investigate 20 cities and a bit less in 2017. And then we also investigate pooled urine samples collected from festivals and at that time we could collect urine from six music festivals in different countries. So we could cover almost all Europe and we could have a first picture of the consumption. We selected some specific substances for target analysis. Here you have the list with different classes of compounds and the most of them were synthetic catenons and phenotylamines. And we also performed the screening analysis. We screened, we selected 200 NPS among those most frequently reported and most recently reported in the early warning systems reports by MCVDA and UNODC in order to have an updated list of the priority NPS at that moment. And as I told you, this has to be done continuously because the market is changing. These are the results from 2016 and 2017. Here I only reported concentration. So we can't really compare the amounts of adjusted profiles between the countries. But we have the most frequently detected where again methadone and made catenone. Methadone is the red one, made catenone which is the green one and also metadone here we have in orange. And more or less the same profile with methadone as the most abundant was found one year later in wastewater. We then tried to investigate to see how the NPS use was comparable with classical industry drugs. Here we have the mass loads. So a milligram per day per 1000 inhabitants. And here we have the mass loads of NPS with the exception of PMA we have loads which are quite low around the one milligram per day per 1000 inhabitants. While if you see here, we have for the classical drugs and especially cocaine we have much higher mass loads. So this means that these substances in the general population are less used than classical drugs. And the similar profile was found in urine from festivals. This is another from another publications. And again, we have concentrations of NPS very low compared for instance to MDMA. Here we have MDMA that in all the festivals investigated is the most abundant substance found in Europe and so used. And the MDMA is another drugs, classical drugs used for recreational purposes. So then we moved to the last project. The last, it means in time because it was run from 2019, 2020, 2022. We revised for this project the list of NPS for target analysis. And we considered eight categories of NPS, 42 substances and also some fentanyl analogs. And we paid a specific attention to include some compounds like TRE, MMC, methyl metcatinone, a an analog of ketamine and the methyl triptamine and the one synthetic opioids isotonitazine because they were in the priority list of EMC-DDA. And so apparently there were substances that may create a treat in that period. We were able to monitor 12 cities in seven European countries with samples collected in 2021. Here we have results. This case we have the mass loads, so milligram per day per 1000 inhabitants. And again, we have mass loads which are around one milligram, so not that high. But here we have a different profile. As you remember, we mostly found in previous studies synthetic ketinone, so only one class of compounds. Here we have for the first time treatments and we have for instance, the methyl triptamine, the blue one, which was found almost in all the cities. And also they compounded it to FDCK was found in several samples. And if you can see here related to the class of synthetic ketinones, before we were finding mostly methadone or other substances, but like a spot analysis. Here we have a trimethylamide ketinone which was found in all the samples and even in quite in higher concentration than the other NPS. And so this means that probably in the last years 3MMC is really the one of the most used NPS in the market. Now 3MMC in several countries is restricted. And so probably we can expect a new substance, one or more will come to the market to replace 3MMC. But we will detect this, so we will monitor this in the next period, in the next years. Finally, I would like to show you also some results from an international network established to monitor the presence of NPS in wastewater leaded by Richard Bade from the University of Queensland in Australia. And Richard asked us, several of us to collect samples during the New Year's Eve in different years. And we performed this since 2019. This is the last publication on this and this referred to the New Year's Eve 2020-2021. And we can see some interesting results. For instance, Utilon was found in New Zealand. It's quite this again, mass loads, you can also compare results. And we have quite high mass loads, especially at the end of the year in New Zealand, less amounts in Australia. And Utilon was not found in Europe, for instance. While on the other side, you can see 3MMC again, and we have it in Europe. It is found commonly in Europe, while it was found in New Zealand, only in a few compounds. So this is very useful and we have seen it in Europe and also in this international study. We can really, with wastewater analysis, provide the differences also in the profile of use in the different countries and over the time. So what we can say is that the first question was if wastewater analysis could be used also for NPS. And I would like to say yes, because we demonstrate with these studies that also despite the challenges we have with NPS, it can be also used as an instrument to see which are the substances used and how the use is changing. We can give a qualitative and quantitative profile of use. We can confirm also from wastewater analysis that the levels of use of NPS in the population is lower than the classical drugs. And it was interesting to see how the pattern was different in different countries and also the changing profiles of use. For instance, methadone, which that is not any more used now and additional substances like 3MMC which are coming in use and which are widely used. So we can say that wastewater-based epidemiology can be a good complementary tool. And even as Joao was telling in the beginning, an indicator, the additional indicator of use of NPS, of course, for NPS and for the other drugs, this information may be then merged with the other epidemiological indicators, the existing one and to have a comprehensive profile, a comprehensive figure. I would like to thank the SCORE Network, of course, first of all, because they provide the samples and the support for doing these projects. European Commissioner, because it's founded in relevant projects and the EMCBDA because they are supporting us in these studies during all these years. And thank you for your attention. Thank you so much, Sarah, for the presentation. And I think it does bring some light for the future on how we can measure NPS use, because as I said before, using traditional service, we know for a fact, and it's also our experience that there are many limitations and it's very difficult to use the traditional service that we have been developing for quite some time now to tackle and disentangle all these new substances that come and go in the European market. So I think wastewater will be quite helpful to establish another tool to measure NPS use. Now it's time for the questions from the audience. I will just say something. There were several comments in the chat asking for the slides. We as a rule, traditional, do not share the slides from the speakers, but the webinar is being recorded and will be made available on our YouTube channel in the coming weeks. So you will be able to go back to the talks that you were interested in. I will now move to the Q&A. I will just read the first questions we have and I will try to point to the right speaker if I can. So I think the first one is actually for Fred. Would it be possible to share the link dashboard report of COVID-19 monitoring through wastewater in the Netherlands? Yes, sure. I will type it in the answer. It's from the Institute of Public Health of the Netherlands but I will put the link in the answer. Thanks Fred. I will also do some publicity because we have been working quite a lot together on the impact of COVID-19 on drug use. So we can also go to the EMCD website where we have specific publications covering also that. 14, do you have any data on the levels of cocaine-specific contaminants, levels specially during lockdown? Some intelligence that due to economic, that due to lockdown, users might be used less reliable dealers with higher levels of bulking as traveling to user dealers may have been more visible to police. So yeah, during the lockdown phases we saw different spatial patterns in the use of cocaine in different European countries. For some of them the use of cocaine remains stable while for others we noticed a decrease in cocaine consumption during this phase. But it's also dependent on which policy changes were made in these countries and to which extent they influenced specific communities in all of these countries. So it's a very heterogeneous question and really location specific. But for Belgium we saw that the use of cocaine did not decrease during the lockdown compared to the period before the start of the lockdown. Thanks, Tim. Just as a compliment, while doing internally some studies on the impact of COVID-19, we also looked at data from drug checking services on the purity and the presence of adulterants in cocaine samples being submitted to drug checking services. And we saw indeed that there was the presence of cocaine samples with more adulterants and less pure than compared with a period before COVID-19. So this from drug checking services, that's what we got. Although the number of samples being submitted decreased quite a lot because of the pandemic restrictions. Another question, are there plans to analyze how a variety of social and economic changes are associated with subsequent changes in drug use patterns shown in wastewater? I maybe can try to give an answer to that but I don't want to steal if the others. There is actually at least one research that I'm aware of and that was done by our colleagues in Australia, the same group that Sarah mentioned. And they did look into relationship within between a whole range of substances that they measured in wastewater and socioeconomic status of different areas that they sampled in Australia. They did, they could find some interesting relationships but from my knowing, it's still very exploratory and at an early stage. And I don't know if you guys, Tim, Sarah know of other studies, but I believe that's probably the only one that has been done so thoroughly at least in that field. There is also one study in Greece that investigated the use of different drugs and pharmaceuticals after the economic downturn. True, we are. Yeah, right, yeah. But if you want, sorry, sorry. Yeah, it's also dependent on the class of compounds but it's interesting that wastewater can be used to analyze these changes. Yeah, if I can add, in Italy, we investigated between 2008 and 2009 with the economic crisis. We found a relevant decrease of cocaine consumption, for instance, and related to the economic crisis. So this was one result that we spotted with wastewater and one year later, also the surveys had the same information. But this was just a spot study. Thank you. Just a quick comment because in the chat, still because of the having access to the slides, people are asking for your email addresses because of data protection issues. I will leave up to you to decide if you want to put your own email address in the chat for the participants or not because they would like to have the slides afterwards. Next question, do you think there is room for the use of artificial intelligence algorithms to help with real-time analysis of wastewater trends? Are you aware of any work being done on this so far? Well, AI and machine learning are being used, but I would not say yet for real-time monitoring. The big bottleneck is that the instruments that we use to detect either the virus or drugs are mostly offline. So they require the sample to be collected throughout the lab analyzed. That's the one. There are efforts to do some of these things online and eventually that might become possible where it's being used as mainly towards the interpretation of the data once it's being collected so that you apply various types of well, not necessarily machine learning or AI, but just regression analysis or multivariate statistics to try to interpret the data and do some more inference on it. And for optimization, I've seen applications to study, to determine which are locations that are better to be sampled and how to sample them based on the structure of the sewer network so that you do like simulations to see how quickly and where would you better collect samples if you want to know what's happening in a certain area. And so what this was done in particular for COVID-19, that's what I am aware of at least. Thanks, Fred. I think this one goes to Sarah. Are there methodological differences in the detection and quantification of synthetic cannabinoids and synthetic catenones in wastewater? For example, are synthetic cannabinoids harder to detect or quantify? No, the methodology is not that different. Is it the same? But the problem is with synthetic cannabinoids is that they are highly metabolized in human body. So you have, we are not finding them mostly because the level of use is low, they are metabolized. And so you are expecting several different metabolites. So it's very difficult to catch them in wastewater. For synthetic catenones, the situation is different because they are less metabolized. And so you can catch the main compound or even some metabolites. So the difficulty with synthetic cannabinoids is that because in wastewater we are expecting much less concentration than urine, of course. And so this is the main challenge with them. Thanks, Sarah. And now I would like to take one from the chat because it's a classic question we all get when presenting or talking about wastewater is the one, how can we distinguish if it's dumping our drugs in the system or if it's used? Can each one of you say something about it? I can try, maybe, no? So ideally we are focusing on human metabolites and there are several criteria requirements to choose a biomarker or a metabolite that we are going to investigate. So human metabolites are coming from consumption and not from dumping. While if you are only analyzing the parent substance in that case, you can have a dumping. And maybe Fred can tell us something about how they spotted the dumping in the Netherlands because they have several examples. Yeah, indeed, in the Netherlands, so as probably some of you know is known, well, actually Belgium as well. Both countries are known for being hotspots of manufacturing of stimulants, in particular amphetamine, MDMA, and recently also methamphetamine are being manufactured in the Netherlands and in Belgium. Well, methamphetamine, I don't know if in Belgium as well, but definitely in the Netherlands. And the thing is that when they manufacture these they create a lot of waste. And the waste also still obviously contains, well, calling it traces is maybe not the right word, quite some amount of the compound that they're trying to synthesize. And sometimes they do get rid of these waste by dumping it in the sewers. Other times they just dump it in nature. But that obviously can cause an increase in the concentration of what we measure. And for amphetamine, MDMA and methamphetamine we do not have a metabolite. So we do measure the substance itself. There are some tricks to detect if something has been dumped. But normally in our case, the dump is so substantial that it's obvious that something was happening because if you would try to estimate how much was being consumed by the population based on how much you measure in waste water it's just a realistic. So you detect it from that. And otherwise there are some things that we can do analytically, it's a bit technical but there are also solutions to do that. So we have ways of seeing it and to a certain extent adjust for it. I can also add that some additional tricks is when the MCD we get the data, internally we start looking at the results from previous years and if we do see a peak for a specific city or for a specific day in a city that looks weird, we do go back to the researchers and score to see that might be a signal of dumping. And in some cases we have decided not to use specific data points because of suspicion. So it's something that we are aware of and that we pay special attention also when reporting the data. So thanks Fred and Sarah for clarifying this. Another question for screening of NPS which libraries are you using and how do you ensure that they kept up to date? Yeah, libraries are, there are few libraries in reality but now they are available and so you can screen with the available libraries. In the beginning where we started we were taking just the few information that was at the time. But now the information is increasing so you can screen and you can have libraries for comparison and you can, it's much better. And since you are on the spot I will send you another question. Regarding the 21 wastewater data are the results directly comparable with the 2017 study? Can you comment on methadone results in 21? Any relationship between results of methadone and three MMC? Yeah, they are simply in 2021 we haven't found the methadone. So the results are comparable. I just presented in different slides and different one was a concentration and the other one was mass load but of course they can be comparable because we are using the mass load related to the population. And what was interesting is that while methadone was found in 2016, 2014 and 15 later now it is not anymore found. And we have a three MMC that probably was replacing it in some way methadone. Of course maybe sometimes you have one compound one molecular to replace a previous one but sometimes you have more than one. So it is difficult also to identify the correct one. Three MMC was the correct one I guess because it was really increasing and this is quite the widespread now. Thanks, Sade. And now actually quite a... I find it very interesting question because it relates to the work we do as well. Are there any attempts to estimate the prevalence of using the population using wastewater data as comparable to surveys and other indirect estimations? We have been... I think it's a bit the holy grail if I can call it like that. Yeah, there have been some attempts using different sources of data I would say not only based on wastewater because you won't be able to do that. You need... Well, in general, you know, wastewater should be always put in context of other indicators not be used alone. But in this case, in particular, if you want to do it, you need really to combine other sources of data and you have to do quite some assumptions based on what you expect one person to consume. You make maybe different categories of users and then you have to take into account all the different uncertainties that are related to purity of the substance and then the wastewater network itself and so on. So there are a few mathematical frameworks that have been proposed to do that. But it's challenging. And the question is, would you necessarily need... Do you really want to do that in a sense that wastewater offers already, in my opinion at least, quite a lot of information without going to that step of trying to estimate prevalence? I guess one of its great advantages is really following trends and so on rather than estimating prevalence. I think maybe there are indicators that are more prone. We're better for that. But that's very personal. Thanks, Fred. If I can use EMCDA jargon, it's very challenging and complex to do that. So I know we have been discussing quite a lot this topic and I think it will be on the table for quite some time. We reached the time of our webinar. I'm really sorry that we were not able to get an answer to all your questions and apologies for that. And also apologies from Alexis, who will not be able to close this webinar. Just from my side, I would like to thank to all the speakers and the colleagues helping organizing it. And I'll pass the floor to Monica just to finish the webinar. So thanks a lot from my side. Thank you very much. We can send the questions, the further questions remain directly to your email address. I think it's quite a meaningful thing that Alexis is not able to conclude this time because in fact, there is no conclusions. I think it's just our first step into this very interesting. We see the reactions from the public. So there will be many other occasions to discuss further about this interesting source of information. Thank you, everybody. Marco will launch a poll to our audience to know how to improve our webinars. You are not required to remain. You have reached your right to go having lunch. And I really thank Alessandra and João for the idea of this interesting topic. Thank you, everybody.