As described on our landing page and project description page the Dutch nitrogen crisis is a complicated problem, with various needs opposing each other (environmental, economical and food-security) and different actors who have very different interests. Finding a fitting solution to the Dutch nitrogen crisis is therefore no easy task, considering that not only does the problem consist of multiple facets, but also the people affected by the problem and possible solutions consist of multiple diverse groups.
It is therefore important that anyone, including us, who is trying to contribute to a solution to this problem, studies the problem and the affected groups in both deepening and broadening research. Especially for (starting) scientists it is important that they step out of the 'ivory tower', and look at the problem they are tackling from outside the lab: it is important that they actively engage with how and IF their solution would work in 'the real world', outside the lab. Only then can a solution be developed that actually addresses the problem; that is actually wanted by and feasible for the people involved in the problem and the solution; and that is responsible, good and safe for use outside the lab.
Throughout the iGEM season, our team has therefore actively engaged in exploring both the problem, and the actors involved in the problem and possible solutions. We have looked at various ethical, economic, environmental, safety and sustainability aspects of our project: not only by consulting literature, but also by actively engaging with people in the field. In addition, over the span of several months, we completed an extensive brainstorming tool to gain more insight into the needs and problems, safety, future, stakeholders and alternatives for our project. Moreover, to ensure the safety of our project even further, we have included several Safe-by-Design principles in our project. Moreover, we created templates that future iGEM teams can use in their Human Practices work to responsibly engage with stakeholders and be more environmentally friendly in the lab. And lastly, we have collaborated with other iGEM teams on their Human Practices work. All to ensure that BYE-MONIA is responsible, good and safe for everyone.
On this Human Practices page, we talk about all the different things we did to research whether BYE-MONIA is responsible, safe and good for the world. On our Integrated Human Practices page, we talk about how we then used this information to adjust our project so that BYE-MONIA is actually responsible, safe and good for the world. All leading to us proposing to use BYE-MONIA as described on our implementation page.
Scope of the problem
By consulting literature we have tried to get a first indication of the scope of the Dutch nitrogen crisis and do in depth research on the Dutch nitrogen crisis. Before work can be done on a solution to the problem, it must be understood how big the problem actually is and whether the problem is actually a problem. A first summary of the results of this research can be found on our project description page. On this page we will discuss the problem in more detail.
The first step of our background research was to investigate which nitrogen-containing compounds are important in the Dutch nitrogen crisis and whether the emissions of these compounds are and have always been too high.
We soon found that, as stated in the project description page, the Netherlands emits too many nitrogen-containing compounds, and that this can be harmful to both people and the environment,,. And although the Netherlands has succeeded in reducing these emissions substantially over the past few years, the decline in emissions has stagnated around 2013, see Figure 1.
There are two (classes of) nitrogen-containing compounds that are part of the nitrogen crisis: ammonia (NH3) and nitrogen oxides (NOx). Nitrogen oxides emissions include emissions of nitrogen monoxide (NO) and nitrogen dioxide (NO2). As shown in Figure 2, ammonia emissions nearly completely originate from agriculture and the decline in emissions has stagnated around 2010. In outdoor air, the ammonia concentration is almost never high enough to be harmful to human health. As shown on Figure 3, nitrogen oxide emissions originate mainly from traffic and emissions continue to decline. When the concentration of nitrogen oxides in the air is too high, it is harmful to human health. People with lung problems and asthma are particularly affected.
We then used literature to investigate why these nitrogen-containing compounds, or rather their deposition, is so harmful to nature and if there is a difference between the deposition of ammonia and nitrogen oxides.
We found that, as described on the project description page, excess deposition of ammonia in nitrogen-sensitive nature areas can harm biodiversity and the quality of habitats in these areas through acidification and eutrophication . When nitrogen oxides deposit in excess in nitrogen-sensitive nature areas, they can also have adverse effects on these nature areas. However, deposition of nitrogen oxides varies a bit from deposition of ammonia. As can be seen in Figure 4, most of the nitrogen deposition in the Netherlands is due to the deposition of ammonia, in the form of reduced nitrogen. Moreover, the acidifying effect of ammonia deposition is almost triple the acidifying effect of nitrogen oxide deposition: The statistical office of the European Union (Eurostat) expresses various air pollutants in equivalents of another air pollutant and ammonia is expressed in 1.9 SO2 equivalents, whereas NOx is expressed in 0.7 SO2 equivalents. Therefore, the total Dutch deposition of ammonia has a bigger eutrophying and acidifying effect than the total Dutch deposition of nitrogen oxides.
We also learned that the limit above which there is a risk that the quality of the habitat will significantly be affected by the acidifying and/or eutrophication influence of (atmospheric) nitrogen deposition is called the “critical load for nitrogen deposition”. To put the Dutch nitrogen crisis in perspective, in 2016, 70%of nature areas in the Netherlands exceeded limits for nitrogen. Moreover, during the 2016-2019 period, 13% of the Dutch water bodies reported poor quality and exceeded the nitrate concentration norm.
Nitrogen deposition in Natura 2000 areas
During our literature review, we noticed that Natura 2000 areas were often mentioned in the discussion of the nitrogen crisis. We therefore paid extra attention to finding out what these areas are and what excess nitrogen deposition means for these areas.
We learned that Natura 2000 is “the largest coordinated network of protected areas in the world”. Natura 2000 areas therefore form “a network of core breeding and resting sites for rare and threatened species, and some rare natural habitat types which are protected in their own right”. The Natura 2000 network stretches across all 27 countries within the European Union and covers areas both on land and at sea. The aim of the network is to ensure the long-term survival of Europe's most valuable and threatened species and habitats. As of December 2020, 15% of the land area of the Netherlands belongs to the Natura 2000 network (EU average: 17.46%). From the 160 Dutch Natura2000 areas (in 2018), 118 were considered to be nitrogen-sensitive areas, based on the presence of at least one nitrogen-sensitive habitat type or the nitrogen-sensitive habitat for species. The European Commission therefore stated that “The high deposition of nitrogen in Natura 2000 areas (above the critical deposition value) requires further efforts in order to protect and restore biodiversity in nature reserves and on farmland”.
NEC guidelines for nitrogen emissions
After investigating which nitrogen-containing compounds are harmful to nature and why, we examined how much of these compounds is actually too much. In other words, we looked into if guidelines have been established on how much ammonia and nitrogen oxides can be emitted.
We found that, to counteract the harmful effects of nitrogen emissions, international emission ceilings have been set for how much each country can emit. The emission ceilings (National Emission Ceilings (NEC)) set for 2010 were intended to reduce the area of Europe affected by acidification by at least half and to reduce the ozone load (which can be formed from NOx emissions) for humans. In 2016 new NEC guidelines were published for 2020. These guidelines are based on agreements made in the Gothenburg Protocol, which applies (at least) to the member states of the European Union, Switzerland, Norway, Croatia and Belarus.
In 2010, the Netherlands was allowed to emit a maximum of 128 kilotons of ammonia according to the Dutch NEC. Of this, 122 kilotons were actually emitted, therefore achieving the 2010 NEC. In 2020, the Netherlands was allowed to emit a maximum of 123 kilotons of ammonia according to the Gothenburg ceiling. It is not yet known whether this ceiling has been met, since the most recent numbers on Dutch ammonia emissions are from 2019. However, if planned policy were to be followed the Netherlands would theoretically have emitted 119 kilotons of ammonia in 2020, thus also meeting the 2020 Gothenburg ceiling . However, as can be seen in Figure 6, total Dutch ammonia emissions have increased again since (approximately) 2013. As a result, the 2010 Dutch NEC was no longer met in 2019, and more action is needed to initiate a decline in ammonia emissions to meet the 2020 Gothenburg ceiling. The European commission then also stated that: “The Netherlands has been found to be at high risk of non-compliance with the ammonia emission reduction commitments for both 2020-2029 and for 2030 and beyond.
Nitrogen emissions from agriculture
Our next step was to examine whether it is correct and fair that agriculture is often mentioned in the discussion of the nitrogen crisis. More specifically, is it indeed agriculture that causes most of the ammonia emissions? And how big is the share of ammonia emissions caused by cows?
We discovered that, as can also be seen in Figure 7, most of the Dutch ammonia emissions indeed originate from Agriculture. Within Dutch agriculture, most ammonia emissions originate from farmed animals, more specifically cattle, as can be seen in Figure 7. In 2019, the Netherlands emitted 125.8 kilotons of ammonia in total, of which 85.45% (107.5 kilotons) originated from agriculture alone. From the total Dutch ammonia emissions, 74.55% (93.79 kilotons) originated from farmed animals and 49.32% (62.04 kilotons) originated from cattle alone .
Moreover, as can be seen in Figure 8, in the entirety of the 27 member states of the European Union, Agriculture, forestry and fishing are the main cause of ammonia emissions (in SO2 equivalents).
Comparing the Netherlands
Since we ourselves knew the nitrogen crisis only as a Dutch problem, we decided to investigate whether this was the case. We therefore used the literature to investigate how much nitrogen and ammonia other European countries emit and how many natural areas they had that were vulnerable to excessive nitrogen deposition (using Natura 2000 areas as a yardstick). At the end we compared this with the same data from the Netherlands. The most important data we found is displayed in Figure 9, which is available (with even more data) in PDF format for download in the link below Figure 9.
Our main conclusions were that, when comparing the Netherlands to other European countries, the Dutch nitrogen surplus is four times the average of the European Union . Moreover, as can be seen in Figure 9, the Netherlands has the second highest ammonia production per square kilometer of land area compared to other (partially) European countries. Moreover, when comparing the ammonia production per square kilometer of each country to the surface area of the country that is covered by Natura 2000 areas (which are, as explained above, generally sensitive to nitrogen), the Netherlands is still in the top 3 of several (partially) European countries. However, it is important to mention that this benchmark is not perfect for comparing the severity of the situation in different countries, considering that also other nature areas can be sensitive to excess ammonia deposition; that ammonia emitted by foreign countries can also be deposited in national Natura 2000 areas and that not necessarily all emitted ammonia will deposit in the national Natura 2000 areas. Lastly, it is notable that ammonia production per capita is not significantly higher in the Netherlands compared to other (partially) European countries.
Downsides of curbing Dutch agriculture
Finally, once it was clear to us that ammonia emissions from farms (and particularly from cows) do indeed pose significant risks to the habitat quality and biodiversity of nitrogen-sensitive natural areas, we decided to see if a much-mentioned solution to this problem was feasible. More specifically, we looked at the potential disadvantages of curbing or even halving the Dutch livestock industry.
We found that, as described on the project description page, curbing Dutch agriculture as the main solution to the nitrogen crisis is not as easy as it might seem. Although it would directly reduce ammonia production by agriculture, thereby reducing the pressure on sensitive natural areas, this solution has many other drawbacks. The Netherlands is the second largest agricultural exporter in the world, and therefore plays an important role in global food security. The European Commission even characterizes the Dutch agricultural industry as “productive, innovative and exportoriented”. Moreover, the European Commission states that the Dutch agricultural industry is “largely based on cost-price reduction and increasing economies of scale” and that it is “very competitive globally, with high labour productivity and a positive trade balance in agrifood products”. Given that the Dutch agriculture industry is internationally cost and product effective, this means that if the Dutch livestock industry is curtailed, and the shortfall in production has to be met elsewhere, it is likely that this (replacement) production will be less effective than Dutch production and thus more raw materials will have to be used for the same amount of production while food prices may also rise. Moreover, curbing this large livestock industry affects the income and job security of the people who work in this industry and thus also affects the gross national product of the Netherlands. In 2019, the production value of Dutch animal agriculture alone was 11.032 billion euros . And lastly, even though Dutch agriculture is responsible for 46% of the nitrogen deposition throughout the Netherlands, nitrogen emissions from abroad are responsible for 32% of the nitrogen deposition throughout the Netherlands. So even if Dutch agriculture is curbed, another major cause of the nitrogen deposition problem is still not being addressed.
Moreover, the proposition of curbing or even halving the Dutch livestock industry has sparked nationwide outrage among farmers, leading to nation-wide farmers protests . Since the exact reasons and motivations behind the outrage are diverse, literature might not be the most suited place to get to know more about the emotional and social background of this outrage. Therefore, we talked to multiple stakeholders, all involved in the nitrogen crisis on an almost daily basis: a farmer, an ecologist and two scientists involved in measuring and modeling nitrogen/ammonia emissions. Their point of view on the subject can be found in the summaries of our stakeholder interviews on our integrated Human Practices page.
Human Practices tools
The nitrogen crisis in the Netherlands is a complex crisis in which more interests, problems and alternatives can emerge than one could imagine at first glance. Therefore, our team decided to do not only in-depth research, but also broadening research in order to understand our project in a broader perspective. To do this, our team made use of two different brainstorming tools: a stakeholder analysis and the WAIR tool.
To identify who is involved in the nitrogen crisis and in our project and to what extent they are involved, our team completed a stakeholder analysis. The idea behind the analysis is to first identify which different stakeholders are involved in the project and which "group" they belong to. Then, for each stakeholder it is reasoned to what extent their lives can be affected by the project (interest / availability) and to what extent they can ensure that the project does or does not go ahead (influence). Finally, the various stakeholders are placed in a grid in which the interest / availability increases from left to right, and the influence increases from bottom to top. This grid indicates for each quadrant how one should then deal with the stakeholders from this quadrant. For example: it is wise to actively involve the stakeholders from this quadrant in your project.
From this final grid, conclusions can be drawn about who the most important stakeholders are to take into account in your project and what the relationships in influence and interest are between the various groups. We also filled out this stakeholder analysis in preparation for our stakeholder interviews. For our project we identified the stakeholders as depicted in Figure 10, and placed them in the grid as depicted in Figure 11.
What is immediately noticeable from the stakeholder analysis is that especially the government (pink post-its) can have a lot of influence on our project, sometimes even more than possible owners (yellow post-its) of the final project. It is also striking that, besides livestock, especially nature (both animals and plants) and future generations have little influence on the problem and the project, while they are precisely the groups that are (or could be) most disadvantaged by the nitrogen crisis.
To see the nitrogen crisis and our solution in a broader perspective, we used the WAIR tool. The WAIR tool is a brainstorming tool created by the Athena Institute and commissioned by the Dutch Ministry of Infrastructure and Water Management. The goal of the WAIR tool is to help young biotechnologists anticipate how their project can interact with the world. It helps young biotechnologists to incorporate socio-technological aspects into the design of their project in such a way that the development of their project meets societal needs and does so in a responsible manner.
Using the WAIR tool, we explored the needs and problems our project addresses or causes; the safety of our project; the alternatives to our project; the stakeholders involved in our project and any future developments that may affect our project.
In order to learn as much as possible from the WAIR tool and to use input from the WAIR tool during different phases of our project, we divided the completion of the WAIR tool over several months. In total, we worked on the WAIR tool almost weekly from the beginning of July until the end of September. We then used insights from the WAIR tool to further develop our project and to be able to ask more specific questions in stakeholder interviews, but we also brought new insights from stakeholder interviews back into the WAIR tool. In this way, we moved back and forth between brainstorming and project development in the real world.
Our completed WAIR tool can be seen in Figure 12.
A stakeholder-based approach
To ensure that our problem actually addresses the needs of the end users and is responsible and safe for use in the real world, we decided to step outside the lab/office and base much of our Human Practices approach on the stakeholders involved in our project. In order to do this, we conducted interviews with stakeholders to learn more about their opinions on the problem and (possible) solutions.
On this page we explain what our process was from beginning to end when contacting stakeholders and which steps we took to ensure that we reached out in a responsible way. The results of the interviews and how they contributed to our project can be found on our integrated Human Practices page.
Contacting stakeholders: beginning to end
Before being able to reach out to stakeholders, we made some preparatory decisions on who to contact and what the goal of the interviews would be. After making the stakeholder analysis, we decided to mostly focus on reaching out to the stakeholders that are in the quadrant of "actively engage". Moreover, even though the 8 months of the iGEM season might feel long, it was not enough to reach out to all the stakeholders that we hoped to reach. We therefore decided to focus on achieving the highest quality of each interview, over achieving the highest quantity of interviews. In addition, we decided that the main goal of the interviews would be to get to know the opinions of different stakeholders on: the (size of) the nitrogen crisis, possible solutions to the nitrogen crisis, the feasibility of our project as a solution to the nitrogen crisis and any possible risks/negative consequences in case our project would be implemented in “the real world”. Lastly, we set up an entire plan to ensure data protection and informed consent during our entire Human Practices work. More about this can be found under “Reaching out in a responsible way”.
After the preparatory work, we followed a step-by-step plan when contacting stakeholders, which is summarized in Figure 13. The entire plan had the goal of getting as much information as possible out of each interview and keeping stakeholders updated in an open way on what was going to happen.
We first contacted stakeholders by telling them: who we are; what iGEM and Human Practices entails; why we contacted them specifically and what we would like to have a conversation with them about. The email script we used for stakeholders we did not know beforehand can be found in Figure 14.
If the stakeholder agreed to meet with us, we would plan a meeting (while keeping the current COVID-19 measures in mind). Before the meeting we would send the stakeholder a list of questions we wanted to ask during the interview - so they had time to prepare beforehand - and our information sheet and informed consent sheet. The information sheet and informed consent sheet we drafted can be found in Figure 15, with important storage places blacked out for safety reasons. Our information sheet and informed consent sheet was partly based on the one from iGEM Exeter 2019.
During the interview itself we would use the questions we send the stakeholder beforehand to have a semi structured interview. During the interview we only introduced our project after we asked the stakeholders about how big they think the Dutch nitrogen crisis is and what a possible solution could be, so as not to bias any answers to those questions. After introducing our project, we would ask the stakeholders about their opinions on our projects and any things we might have overlooked. Even though, as stated in the information and informed consent sheet, we expected our interviews to take 30-60 minutes, they often took around 60-90 minutes (if the stakeholder agreed to that as well).
After the interview, we would use any recordings and notes we had of the interview to write a summary of the interview. These summaries were all written according to the AREA-framework as introduced to iGEM by iGEM Exeter 2019. As iGEM Exeter 2019 writes: “The AREA Framework for Responsible Innovation, created by Professor Richard Owen seeks to promote creativity and opportunities for science and innovation that are socially desirable and undertaken in the public interest and is used by many research councils to encourage a responsible approach to innovation”. A graphical summary of the 4 steps of the AREA framework can be found in Figure 16. By using the AREA-framework to structure our summaries we tried to not only reuse the valuable information provided by previous iGEM teams, but we even wanted to build upon it! Seeing that we decided to focus on quality over quantity, we decided to fill in more elaborate frameworks than iGEM Exeter 2019 did. This also came in useful considering that most of our interviews ended up being 60-90 long, so there was a lot of valuable information that could more effectively be summarized by using the AREA framework. Before the summaries were posted, consent from the stakeholder was asked to publish that specific summary on a specific place (either the wiki or newsletter).
After the interview, we offered the stakeholder the opportunity to receive our monthly newsletter. The purpose of this newsletter was to keep our stakeholders updated on the progress of our project and how their input helped us move our project forward. Or in other words, the newsletter was a form of "aftercare" to ensure that the stakeholder was not left in the dark after the interview but had the opportunity to stay involved in the project. An example of one of our newsletters can be found in Figure 17. We did include summaries of two stakeholder interviews in this specific newsletter, but those are not included in Figure 17 to keep Figure 17 readable, but can instead be found on our integrated Human Practices page.
Reaching out in a responsible way
Talking to stakeholders is a good way to develop a safer, more appropriate and more complete iGEM project, but it can also involve risks. Stakeholders are people and therefore -of course- have rights, feelings and personal data that should be handled responsibly. If these are not handled responsibly, the team might be at risk of unethical and data-security breaching behaviour. In other words: when taking into account the safety of your project, it is important to not only think about the biological safety of your project, but also think about the data-safety issues you might face during your Human Practices work. It is therefore important that every iGEM team, including us, that engages with stakeholders is aware of:
- What data they are collecting
- Why they are collecting this data
- Whether it is really necessary to collect all of the data that is collected
- Whether the stakeholder has given and can give permission to collect this data
- Whether the collected data is personally identifiable
- How long the collected data will be stored
- Where the collected data will be stored
- Who has access to the collected data
- Whether the stored data is sufficiently secure
In short: to reach out (to stakeholders) in a responsible way, it is important to make sure that your project takes the data protection and informed consent of the people you engage with into account. Therefore, our team set up an entire plan to ensure data protection and informed consent during our entire Human Practices work. A graphical summary of this plan can be found in Figure 18.
The first thing we did to ensure that we reached out in a responsible way, was to make decisions on what data we would like to collect and how we would like to store it. The first big decision that we made is that we would avoid asking our stakeholders questions about data with a high safety risk such as their religion, age and seksuality, considering that this data would most likely not help us develop a better solution to the Dutch nitrogen crisis but would be extra sensitive and personal data; so collecting it should be avoided whenever possible.
Moreover, we decided to avoid interviewing vulnerable participants, considering our project topic did not necessarily call for interviewing vulnerable participants but interviewing vulnerable participants does bring additional (ethical) risks for ensuring informed consent. Vulnerable research participants are people for whom the exercise of autonomy is compromised or for whom there is an unequal power relationship between the data subject and the research party. Vulnerable research participants therefore cannot freely give or refuse consent to the processing of their data,. Examples of vulnerable people are prisoners, children and people in critical medical conditions or a coma. The only time that we did work with minors, during the implementation of our education work, we took extra precautions to ensure that either consent from the parents/caretakers was given or only non-personal identifiable and non-copyrighted material was collected.
In addition we looked at the extent to which we wanted to collect personally-identifiable information. Seeing that even information that does not include the name or contact details of a stakeholder, could already be personally identifiable if the story is detailed enough. For example: “a person wearing a red shirt” is not personally identifiable, but “a person wearing a red shirt, living at the Herestraat in Groningen, walking their German Shepard every day at 06:10 AM” could be. We therefore decided to not include names in our internal notes and very clearly ask our stakeholders whether the summary we wrote could include any personally identifiable information or not. Moreover, we asked iGEM HQ if personally identifiable summaries that we published on our wiki - after approval of the stakeholder - could still be made anonymous if the stakeholder so wished, even after the wikifreeze. Luckily, this was possible.
Lastly, we decided to work through trusted and password protected platforms (for emails and video calls) as much as possible and in addition, password protect as much of the data we collected as possible. More specifics about what data we collected, for how long and where we stored it, can be found in Figure 15.
After deciding which data we would like to collect, we checked if, with our current approach, we were still following several different regulations that applied to us. We looked through the Dutch General Data Protection Regulation (GDPR), and we looked into whether we would need to set up a Data-protection-impact assessment (DPIA) and appoint a Data-protection officer (DPO),. We came to the conclusion that with the data that we planned on collecting - which did not include data with a high safety risk nor did it include large-scale data processing - and the people we planned on interviewing - which did not include vulnerable research populations - we were compliant with the Dutch GDPR and did not need to set up a DPIA or appoint a DPO.
All of those decisions led us to create the information sheet and informed consent sheet as displayed in Figure 15. After having written a first draft of the document, we contacted the data-security office of our university and the ethics committee of the faculty of social and behavioral sciences of our university to help us improve the document we had. Moreover, we also asked our supervisors to check the document and supply us with any comments they had. After having the final version of our information sheet and informed consent sheet, and therefore having our framework set up for how we would handle data-collection during our stakeholder interviews, we were confident that our Human Practices work ensured informed consent and data protection of our stakeholders.
One of the most important aspects during the development of your iGEM project, is ensuring the safety of your project. Typically, this means that teams perform their lab work in accordance with applicable safety regulations for microbiological research. Some teams go even further and design their project so that safety risks that may occur when their GMO leaves the lab are mitigated. We also incorporated both of these aspects into our project, as can be read on the safety page.
However, at iGEM Groningen 2021, we also believe that the safety of an iGEM project goes beyond the lab and the possible spread of your GMO into the outside world: equally important is the consideration of whether your project is safe in terms of economics, data protection and environment. For example: Does your project increase economic inequality? Or does your Human Practices approach jeopardize the data security of your stakeholders? Or does the implementation of your project support environmentally-unfriendly choices?
In order to incorporate all of these aspects of safety of our project - from inside the lab, to just outside the lab, to well into society - into the design of our project, we created a comprehensive Safe-by-Design (SbD) approach for our project. SbD is an approach to developing a research project in which the developers aim at “addressing safety issues already during the R&D and design phases of new technologies”. For our SbD approach we made several SbD choices for the design of our project, which will be implemented during specific phases of our project.
We have made the following choices for our project to be developed according to the SbD principle and to be able to mitigate the safety of our project in the broadest sense of the word. Alternatively, the safety-section of our filled-in WAIR tool also contains a good explanation of the SbD decisions we made.
To ensure the biological safety of our project and to mitigate any risks when working with our GMO inside the lab and to mitigate any risks if our GMO would be accidently spread to the outside world, we decided to pick a chassis that is not only easy but also safe to work with. Moreover, this design choice would significantly lower the dual use potential of our GMO. During our project we have used both Saccharomyces cerevisiae and Saccharomyces paradoxus as a host for alpha-amylase production. Neither S. cerevisiae or S. paradoxus has been associated with pathogenicity toward humans or has been shown to have adverse effects on the environment.
To ensure our feed-additive did not cause any safety risks for: the animals that ingest it; the people who work with it; or the environment where it could (accidentally) end up in, we decided to pick a safe feed additive. Moreover, this design choice would significantly lower the dual use potential of our GMO. Since - understandably - within iGEM we could not test our feed additive for cow, human and environmental safety, we decided to dive into the literature. We found that a similar feed additive with alpha-amylase as the active ingredient, was extensively reviewed on these topics by the European Food Safety Authority (EFSA). The study by EFSA concluded that no concerns for consumer safety arose from the use of the feed additive for dairy cows. In addition, the feed additive is not considered to be irritant to human skin or eye. The feed additive is however considered to be a potential skin sensitizer, but only because no data was provided to test for skin sensitization. Moreover, the additive is considered to be a potential respiratory sensitizer because of its proteinaceous nature. However, since the active substance of the feed additive is a protein, it will be degraded/inactivated during passage through the digestive tract of animals. Therefore, the EFSA expected no risks to the environment and concluded that no further environmental risk assessment is required. In addition, in our own project, we chose alpha-amylase genes to clone which’s pH and temperature optimum most closely matched the pH (physiological range: 5.8-6.5) and temperature (around 38-39 degrees ) of the rumen of cattle. In this way, our alpha-amylase would show the most activity in the rumen, but would be quickly degraded in the rest of the cow's digestive system where the pH and temperature is different from that of the rumen.
Although we had already looked at the safety of both the chassis and the enzyme to be expressed, we wanted to build in an additional safety measure to guarantee the safety of our project for people, animals and the environment. We therefore chose to keep the GMO within the confined boundaries of the laboratory/production plant, within which strict rules apply to prevent spread of the GMO. To make this possible, we have chosen to use the GMO to produce the feed additive in the lab. The feed additive will then be purified, so that only the feed additive will leave the lab and be added to the feed of cows. A similar feed additive produced with a GMO  already shows that this is possible and that no traces of the GMO or engineered genes can be found in the purified feed additive. In addition, we have incorporated the MOF (the filter that collects ammonia) into our project so that we can bring ammonia from the outside world to the lab. This means that the GMO does not have to leave the lab, but can still use ammonia "from the outside world".
During our project, we ensured - as also required from iGEM - that all rules for safe microbiological techniques were followed in the lab, in order to ensure the safety of our project for humans, animals and the environment. These rules would ensure that the GMO could not escape from the lab, and that the risks to the people working in the lab were minimized. Although we ourselves have only worked on the project on a laboratory scale so far, these rules should also be followed in industrial production facilities that produce our GMO and feed additive on a large scale in any later stage of the project. The specific safety measures we have followed in the lab and specific courses we have taken can be found on our Safety page.
Despite the fact that other SbD choices are already geared towards ensuring that the GMO is not harmful to the world outside the lab or are geared towards keeping the GMO in the lab as much as possible, there is always a chance that the GMO will still escape from the lab/production plant and then cause harm to the outside world. Especially when working with the GMO on a large scale, the danger after and the risk of human malpractice, spills or other events that could take the GMO outside the lab are significantly higher. Our team has therefore chosen to design a kill switch, as described on our Safety page, that prevents the GMO from surviving without the administration of galactose, which in theory it should not receive outside the lab. As a result, our GMO would not be able to survive outside the lab. This kill switch would mainly be of importance during the industrial production of our feed additive. Moreover, this design choice would significantly lower the dual use potential of our GMO.
From the standpoint of safety of economic equality, we chose to design our project to produce as much feed additive as possible with as few raw materials as possible. Indeed, should our project eventually be implemented on a large scale and would farmers be able to gain a large (economic) benefit from the use of our feed additive, our feed additive might become an important part of the economic competition. Should our feed additive then also be very expensive, so that only richer farmers can afford it, then richer farmers could gain more economic benefit from it than poorer farmers. This could increase income inequality between poorer and richer farmers, both inside and outside the Netherlands. We have therefore chosen to keep the production price of our feed additive as low as possible by looking, with the help of modeling, for the best combination of promoter, signal sequence, gene and Saccharomyces strain, for the most alpha-amylase production with the same amount of raw materials. In addition, we chose our GMO so that it could use ammonia as its only nitrogen source. As a result, as long as the MOF captures enough ammonia, we would not need to provide our GMO with additional nitrogen sources but can reuse a "waste product" from the process.
We wanted to ensure that both the development of our project itself and the final implementation of our project caused as little harm to the environment as possible. Therefore, we have encouraged as many environmentally friendly choices as possible during our project, and also propose to continue these environmentally friendly choices during any implementation of our project. First, we worked paper-free as much as possible during our project: both the stakeholder analysis and the WAIR tool were created online. In addition, we also tried to reuse materials from old iGEM teams in the lab. In addition, we rejected one of our first project ideas in part because it would produce greenhouse gases that are harmful to the environment. And finally, despite not having reached that stage ourselves during our iGEM season, we propose to use reusable energy sources to produce the heat needed to heat up the MOF to release the adsorbed ammonia.
Because we want to be engaged in safety in its broadest sense, we also looked at how developing our project might affect the people with whom we come into contact. To ensure that this was done as ethically as possible, and that it was as clear as possible to both us and our stakeholders what data we were and would be allowed to collect, we paid extensive attention to drafting an information and informed consent sheet. This sheet had the goal of ensuring both the data protection and informed consent of our stakeholders. More specifications on this sheet can be found under "a stakeholder based approach".
The first step that must be taken if one wants to prevent security risks is to understand what risks there may be. Only when one knows what risks may occur can one make design choices to prevent these risks. Our team has therefore focused throughout our iGEM season on understanding: the problem we are addressing; the potential security risks of our chassis and feed additive; the dual-use potential of our project; and the risks our project may pose in the broader context outside the lab, i.e., for food safety, economic equity, and data protection. To understand these risks, opportunities and possible solutions as much as possible, our team conducted extensive literature research; completed extensive brainstorming tools; engaged with various stakeholders and attended several meetups. Although "investigating risks'' may sound like an open door, in reality it is an important step that should not be forgotten and that we therefore consciously included in our SbD approach.
Implementing safe-by-design along the way
During the development of our iGEM project, different project stages can be identified, starting with coming up with a project idea and ending with our project being implemented on farms. During different stages of the project, different SbD choices play a role. Normally, projects start their development with an extensive brainstorm on possible ways of solving the problem and any risks associated with different solutions. After that, most teams move to the lab to provide a proof of concept in which the teams demonstrate on a small scale that their project could actually work and has no major safety risks. Only after that, and probably after even more safety and efficacy testing, could a project be scaled up in industrial production to be used in real life. Most importantly, SbD plays a huge role in preventing the dual use potential of any biotechnology project.
Even though the 8-month long iGEM season is not long enough to go through every project stage, we would still like to (schematically) highlight during what stage of the project we think each of our SbD choices plays a role. The different stages of the project are in chronological order. The last stage mentioned is the stage in which dual-use of the project should be prevented. However, this “last” stage actually works alongside every other stage of the project.
Proof of concept in the lab
Industrial production and real life application in farms
Prevention of dual use of the project
Tools for future iGEM teams
Setting up, designing and running an entire iGEM project is a lot for only 8 months. Especially if teams also need to design and run an entire Human Practices approach besides their lab work and other duties. We ourselves encountered that setting up our Human Practices approach and drafting the needed documents took quite some time, which therefore meant that we had less time to speak to stakeholders. In the end, this meant that we were unable to speak to all the stakeholders that we wanted to speak to. We have therefore designed several flowcharts and a template that could help new iGEM teams design their Human Practices approach, so that they can spend more time on actual stakeholder interviews. When all three of these documents are followed, teams should have set up most of the important aspects to ensure data protection, informed consent and responsible outreach aspects in their stakeholder-based interviews. Moreover, we have designed an experiment/protocol which will help teams be more sustainable in the lab. This will allow teams to develop their project more responsibly by being more environmentally friendly in the lab.
Templates to help design Human Practices approach
The first step in any good Human Practices approach that involves performing stakeholder interviews should always be to look at what data your team aims to collect; where and how long you would like to store it and check if all of this is still in compliance with regulations that apply to your university and country. A simplified flowchart that can be followed to help you navigate the most important questions is displayed in Figure 19.
Once it is clear what data your team would like to collect, it is wise to draft an information and informed consent sheet to send to your stakeholders before the interview. A template based on our information and informed consent sheet can be found in Figure 20. Within the template the yellow-highlighted text can be replaced with the team's own information and the blue-highlighted text are important notes for the teams using the template. This template is mostly suited for teams that would like to follow our Human Practices approach in which notes and recordings are made of the interview, a summary is written afterwards and much of the data is deleted after the iGEM season. However, each team is ofcourse free to adjust the template based on their own preferences. Do be sure to check this information sheet and informed consent sheet with your supervisors, the ethics committee and data-security office of your university and any other relevant organizations/regulations that apply to you before using it!
Once you have drafted and checked your information and informed consent sheet, you can approach stakeholders. We ourselves did so according to the scheme in Figure 21, and we think other teams might be able to re-use this approach. Figure 21 is explained in more detail in the section “A stakeholder based approach”.
Sustainability test for in the lab
When coming to the lab for the first time, we noticed that previous iGEM teams had left quite some materials. However, some of these materials were past the expiration date and we were unsure whether we could still use them or not. We can imagine that, now that iGEM is getting bigger and bigger each year and more universities are returning for the competition each year, that other teams might face the same issue. We have therefore designed and performed a sustainability test which teams can use to test if they can reuse old mini-prep kits so that they do not necessarily need to throw them away. The explanation of this test can be found on our contributions page.
Human Practices collaborations
Aside from doing Human Practices work for our own project, we have also helped other teams with their projects!
We have helped iGEM Wageningen University and Research (WUR) 2021 test and improve their guide for organising a stakeholder meetup simulation, as is described on our collaboration page.
Next to that we have also collaborated with iGEM Patras 2021 to develop a video showcasing all the Sustainable Development Goals that iGEM 2021 teams are working on, as also described on our collaboration page.
Lastly, we sat together with our partner iGEM IISER Tirupati 2021 to record a podcast episode about the synthetic biology in the food industry, as described on our partnership page.
- European Commission, “Commission recommendations for The Netherlands’ CAP strategic plan Accompanying the document COMMUNICATION FROM THE COMMISSION TO THE EUROPEAN PARLIAMENT, THE COUNCIL, THE EUROPEAN ECONOMIC AND SOCIAL COMMITTEE AND THE COMMITTEE OF THE REGIONS Recommendat,” Comm. Staff Work. Doc., 2020.
- Rijksinstituut voor Volksgezondheid en Milieu, “Stikstof | RIVM.” [Online]. Available: https://www.rivm.nl/stikstof. [Accessed: 11-Oct-2021].
- “De Levende Natuur issue 2 of 2013 | Theme: Toekomst voor de natuur,” Levende Nat., 2013.
- “De Levende Natuur issue 3 of 2013 | Theme: Toekomst voor de natuur (part 2),” Levende Nat., 2013.
- Rijksinstituut voor Volksgezondheid en Milieu, “Stikstof - Stikstofoxiden (NOₓ) .” [Online]. Available: https://www.rivm.nl/stikstof/stikstofoxiden-nox. [Accessed: 13-Oct-2021].
- European Environment Agency, “Ammonia (NH3) emissions — European Environment Agency,” 04-Sep-2015. [Online]. Available: https://www.eea.europa.eu/data-and-maps/indicators/eea-32-ammonia-nh3-emissions-1. [Accessed: 29-Aug-2021].
- statistical office of the E. U. Eurostat, “Air emissions accounts by NACE Rev. 2 activity (env_ac_ainah_r2).” [Online]. Available: https://ec.europa.eu/eurostat/cache/metadata/en/env_ac_ainah_r2_esms.htm. [Accessed: 13-Oct-2021].
- H. F. van Dobben, R. Bobbink, D. Bal, and A. van Hinsberg, “Overzicht van kritische depositiewaarden voor stikstof, toegepast op habitattypen en leefgebieden van Natura 2000,” Alterra-rapport 2397, Dec. 2012.
- European Commission, “Natura 2000 - Nature and biodiversity - Environment.” [Online]. Available: https://ec.europa.eu/environment/nature/natura2000/index_en.htm. [Accessed: 11-Oct-2021].
- European Commission, “‘Branding’ Natura 2000goods and services,” Nat. 2000 Newsletter, Nat. Biodivers. Newsl., no. 50, Jul. 2021.
- A. M. Schmidt and R. A. Smidt, “Scientific analysis of the status of designated Natura 2000 areas and the protection of nitrogen-sensitive species and habitats Dutch contribution,” Wageningen Environ. Res., Apr. 2018.
- N. en V. Natura 2000 - Ministerie van Landbouw, “Natura 2000 gebieden .” [Online]. Available: https://www.natura2000.nl/gebieden. [Accessed: 11-Oct-2021].
- Rijkswaterstaat Ministerie van Infrastructuur en Waterstaat, “NEC-stoffen - Kenniscentrum InfoMil.” [Online]. Available: https://www.infomil.nl/onderwerpen/lucht-water/lucht/nec-stoffen/. [Accessed: 29-Aug-2021].
- “Absolute emissiereeks ammonia (in [kg]) naar Lucht 2019,” Emmissieregistratie, Aug-2021. [Online]. Available: http://www.emissieregistratie.nl/erpubliek/erpub/weergave/grafiek.aspx. [Accessed: 21-Aug-2021].
- Eurostat, “Air pollution statistics - air emissions accounts,” Statistics explained, Feb-2020. [Online]. Available: https://ec.europa.eu/eurostat/statistics-explained/index.php?title=Air_pollution_statistics_-_air_emissions_accounts#Acidifying_gases. [Accessed: 13-Oct-2021].
- Eurostat, “Air pollutants by soure sector (source: EEA),” Eurostat air emission inventories (source: EEA) web explorer, 04-Oct-2021. [Online]. Available: https://appsso.eurostat.ec.europa.eu/nui/submitViewTableAction.do. [Accessed: 07-Oct-2021].
- Eurostat, “Area by NUTS 3 region,” Eurostat - Data Explorer, 08-Feb-2021. [Online]. Available: http://appsso.eurostat.ec.europa.eu/nui/submitViewTableAction.do. [Accessed: 07-Oct-2021].
- Eurostat, “Population on 1 January,” Eurostat data browser, 05-Jul-2021. [Online]. Available: https://ec.europa.eu/eurostat/databrowser/view/TPS00001/bookmark/table?lang=en&bookmarkId=c0aa2b16-607c-4429-abb3-a4c8d74f7d1e. [Accessed: 07-Oct-2021].
- European Commission, “The EU pollinators initiative,” Nat. 2000 Nat. Biodivers. Newsl., vol. 44, no. July, 2018.
- M. Dolman, G. Jukema, and P. Ramaekers, “De Nederlandse landbouwexport 2018 in breder perspectief,” Wageningen Econ. Res., Jan. 2019, doi: 10.18174/468099.
- Centraal Bureau voor de Statistiek, “Landbouw droeg in 2019 evenveel bij aan economie als tien jaar eerder,” CBS website - nieuws, 07-May-2020. [Online]. Available: https://www.cbs.nl/nl-nl/nieuws/2020/19/landbouw-droeg-in-2019-evenveel-bij-aan-economie-als-tien-jaar-eerder. [Accessed: 13-Oct-2021].
- E. Stokstad, “Nitrogen crisis threatens Dutch environment—and economy,” Science (80-. )., vol. 366, no. 6470, pp. 1180–1181, Dec. 2019, doi: 10.1126/SCIENCE.366.6470.1180.
- Autoriteit Persoonsgegevens, “Data protection impact assessment (DPIA) .” [Online]. Available: https://autoriteitpersoonsgegevens.nl/nl/zelf-doen/data-protection-impact-assessment-dpia#wat-zijn-de-criteria-van-de-europese-privacytoezichthouders-6668. [Accessed: 13-Oct-2021].
- P. A. M. Gómez, “Nature of Vulnerability in Biomedical and Psychosocial Research,” Clin. Trials Vulnerable Popul., May 2018, doi: 10.5772/INTECHOPEN.70186.
- European Parliament and the counsil of the European Union, “VERORDENING (EU) 2016/ 679 VAN HET EUROPEES PARLEMENT EN DE RAAD - van 27 april 2016 - betreffende de bescherming van natuurlijke personen in verband met de verwerking van persoonsgegevens en betreffende het vrije verkeer van die gegevens en tot intrekking van Richtlijn 95/ 46/ EG (algemene verordening gegevensbescherming),” Off. J. Eur. Union, Apr. 2016.
- Autoriteit Persoonsgegevens, “Algemene verordening gegevensbescherming (AVG) .” [Online]. Available: https://autoriteitpersoonsgegevens.nl/nl/over-privacy/wetten/algemene-verordening-gegevensbescherming-avg. [Accessed: 14-Oct-2021].
- Justitia.nl, “Data Protection Officer (DPO) / Functionaris Gegevensbescherming.” [Online]. Available: https://www.justitia.nl/privacy/data-protection-officer. [Accessed: 14-Oct-2021].
- I. van de Poel and Z. Robaey, “Safe-by-Design: from Safety to Responsibility,” Nanoethics, vol. 11, no. 3, p. 297, Dec. 2017, doi: 10.1007/S11569-017-0301-X.
- United States Environmental Protection Agency, “ATTACHMENT I--FINAL RISK ASSESSMENT OF SACCHAROMYCES CEREVISIAE,” 1997.
- European Food Safety Authority, “Scientific Opinion on the efficacy of Ronozyme® Rumistar (alpha-amylase) as a feed additive for dairy cows,” EFSA J., vol. 11, no. 10, Oct. 2013, doi: 10.2903/J.EFSA.2013.3434.
- “Information on EC 220.127.116.11 - alpha-amylase,” Brenda. [Online]. Available: https://www.brenda-enzymes.org/enzyme.php?ecno=18.104.22.168. [Accessed: 11-Oct-2021].
- J. Dijkstra et al., “Ruminal pH regulation and nutritional consequences of low pH,” Anim. Feed Sci. Technol., vol. 172, no. 1–2, pp. 22–33, Feb. 2012, doi: 10.1016/J.ANIFEEDSCI.2011.12.005.
- N. H. Rutherford, A. W. Gordon, F. O. Lively, and G. Arnott, “The Effect of Behaviour and Diet on the Rumen Temperature of Holstein Bulls,” Anim. an Open Access J. from MDPI, vol. 9, no. 11, Nov. 2019, doi: 10.3390/ANI9111000.
- P. A. Gibney et al., “Common and divergent features of galactose-1-phosphate and fructose-1-phosphate toxicity in yeast,” https://doi.org/10.1091/mbc.E17-11-0666, vol. 29, no. 8, pp. 897–910, Apr. 2018, doi: 10.1091/MBC.E17-11-0666.