Threats from the micro universe

There was a time when everyone was talking about the threat of heavy metals, solvents and oil pollution. That talk is still relevant today, but now we are also to a great extent talking about endocrine disruptors, nanoparticles, microplastics and even nanoplastics.

The technological revolution has created thousands of different tiny molecules and particles that may affect us ... only, scientists don't actually yet know how, because some of these research fields are new: The first studies are no more than just a few years old.

In addition, finding the head and tail of the problem is a very complex matter, and therefore no one really knows the size or extent of the threats. At the same time, new microscopic substances are developed every day, and they end up in the air, water and food; the very basis of life for humanity.

Researchers have seen some clear indications that we need to regulate the industrial sector’s emission of micro- and nano-sized substances. While some of the microscopic substances hold the promise of cutting-edge medical breakthroughs, others threaten to destroy both us and nature from within.

It is up to the researchers to find out which substance does what, and to advise legislators on the next step before it all bolts.

Some of the world’s foremost researchers in the field can be found at University of Southern Denmark. With both large international financial grants and access to some of the most advanced equipment in the world, they are determined to set the direction for the future.

We have interviewed Assistant Professor Elvis Genbo Xu, Professor Frank Kjeldsen, Professor and Head of Department Horst-Günter Rubahn and Associate Professor Henrik Holbech about the present and future of the threats from the micro and nano universe.

Plastic turns into microplastics

You must have been on a completely different planet the last few years if you have not heard of microplastics. The media (and for that matter also the oceans) are full of it.

Microplastic is mainly formed in the ocean, where the movements of the ocean help tear plastic apart like a giant shredder. First, the plastic shopping bag is torn apart. Then the big chunks of plastic are torn into smaller chunks. The smaller pieces are crushed even more until the pieces of plastic are so small that you can no longer see them with the naked eye.

However, they are clearly visible under a microscope, and just within the past year, researchers have concluded that microplastics are everywhere and can be found in every cubic metre of the world's oceans and even buried in the deepest seabed.

These tiny pieces of plastic are absorbed by the animals and plants in the ocean, accumulating in larger and larger quantities, meaning that the tuna steak on your plate could be filled with microplastics from everything from shopping bags to disposable razors and plastic bottles. Enjoy your meal!

You may well ask yourself: ‘Is it really that dangerous?’. The answer is that researchers are still trying to figure it out.

‘Several studies have established that microplastics can affect the survival, growth, food intake and reproduction of different organisms. However, these studies were conducted using well-defined microplastics that are very different from the microplastics found in nature, where there is a large variation in polymer types – shapes, sizes and different surface characteristics. So far, we know very little about this mix of microplastics’, explains Elvis Genbo Xu, an ecotoxicologist and Assistant Professor at the Department of Biology, University of Southern Denmark.

Microplastics turn into nanoplastics

However, the scary perspective doesn't stop at the microplastics. The natural next step, when the plastic takes another turn through the giant shredder, is that the microplastics turn into nanoplastics.

If the researchers are uncertain about the effect of microplastics, they have nothing to go on when it comes to nanoplastic. And there is actually a significant difference between the two.

‘When the plastic becomes nano-sized, the particles can penetrate through cell membranes and into the cells. This is even more frightening because the effect on cells is even more unknown than the effect of microplastics. We have seen in some experiments with primitive animals that nanoplastics can penetrate cells, but we don’t know much about the long-term consequences’, says Elvis Genbo Xu.

He explains that two fields need more research in the future before it becomes possible to make legislation and offer advice based on reason and knowledge.

First, scientists need to find out how much nanoplastic is held in the oceans and in the rest of the environment around us. This in order to understand how much we are exposed to. In fact, we may be exposed to the most in our homes or the office, where particles from many types of furniture and objects around us fill the air. In addition to the techniques for quantifying nanoplastic in the environment, researchers also need to develop some techniques to differentiate between nanoplastic and biological molecules found in, for example, a cell.

Second, which is Elvis Genbo Xu's primary research area, scientists also need to find out what it means to you, me and everyone else that our cells are invaded by nanoplastic. These kinds of investigations start with cell-level experiments where scientists expose cells to different doses of nanoplastic and see how they respond. Subsequently, the experiments are moved to animals to see if the cell-level effects are expressed at organism level.

A whole third layer of the problem is that plastic as a material is very good at absorbing other substances, such as endocrine disruptors, which in this way can gain free access to the cells and subsequently be released.

‘Some tests on frogs and daphnia show that even large amounts of nanoplastic are not deadly, but there can still be some negative effects over time that we have not yet elucidated’, says Elvis Genbo Xu.

Endocrine disruptors harm both humans and the environment

Endocrine disruptors is the research area that interests Henrik Holbech the most. He is an ecotoxicologist and Associate Professor at the Department of Biology, University of Southern Denmark.

They can be man-made chemicals, but they can also be naturally created substances. If we need to make a quick definition, endocrine disruptors are extraneous molecules that can affect the hormone systems in the body and thus harm the organism.

The problems with the endocrine disruptors apply to our health as well as to the environment:

In the environment, the endocrine disruptors can change, for example, the relationship between males and females of a given population of animals. In studies of endocrine disruptors in the laboratory, researchers such as Henrik Holbech use, among other things, zebrafish to see what amounts of a substance are needed before the consequences become serious for the gender distribution.

When concentrations become too high, you see more males than females, and if you transfer that conclusion to nature, it is not difficult to see how endocrine disruptors in the environment can push the delicate natural balance between the sexes and perhaps push some species to the edge of extinction.

‘A classic example is ethinylestradiol, which is an estrogen. It was previously used in birth control pills and via them released to the environment. Males are not intended to be exposed to great amounts of estrogen in our environment, and males from different animal species can be seriously affected’, explains Henrik Holbech.

In the human body, the endocrine disruptors can also have serious consequences. They may, among other things, affect the onset of puberty, but they can also affect metabolism and thus possibly lead to obesity or type 2 diabetes.

‘Especially during fetal development, humans are very vulnerable to endocrine disruptors because the entire fetal formation is orchestrated very precisely by the hormones, among other things. Therefore, changes in hormone balance may affect normal fetal development. The most extreme example of the external hormones is that if you want to go from being a man to a woman or vice versa, you consume large amounts of sex hormones, and that changes both the body and the psyche’, says Henrik Holbech.

Denmark has been at the forefront of this development

In the laboratory at the Department of Biology at SDU, Henrik Holbech and colleagues work with several different types of endocrine disruptors to investigate their effects on different organisms such as fish, toads and invertebrates. The researchers are investigating, among other things, the effects on metabolic hormones and androgens, which are the male sex hormones.

Specifically, Henrik Holbech received a large EU grant (Horizon 2020) to develop, together with 15 partners in 8 countries, better test guidelines to test whether chemicals are endocrine disruptors.

There are different perspectives in this research area.

The negative

The effect of endocrine disruptors is real. The environment has seen severe effects on populations of snails, reptiles, birds and fish, and if we look at humans, Denmark has the world's second highest incidence of testicular cancer among men, and for about 10 percent of all newborn boys, their testicles have not fallen into the scrotum, as they should. Both can probably be credited with the fact that during their development Danish men and boys are exposed to substances that alters their natural hormone balance.

Add to that, the EU alone has approved more than 20,000 industrial chemicals that have not yet been tested for their potential endocrine disrupting properties. So far, researchers have only examined a few hundred.

‘The problem is that when you approve a new industrial chemical, you have to prove that it is not toxic or carcinogenic, but there is no requirement to prove that it is not endocrine disrupting’, explains Henrik Holbech.

The positive

In Denmark, we are very good at cleaning our wastewater, which means that the problem of endocrine disruptors in our environment and in nature is much less today than it was, say, 20 years ago, when we saw effects on some of our freshwater fish. On the plus side, too, is that the world - and especially the EU - is facing the problem. This means that large sums of money are being allocated to researchers like Henrik Holbech, who have set out to do something about it.

‘Internationally, the problem of endocrine disruptors in the environment is greater than in Denmark, because in many places in the world, wastewater is not cleaned’, says Henrik Holbech.

Test methods for the identification of endocrine disruptors

Henrik Holbech's work consists of developing test methods that make it possible to quickly determine if a chemical is endocrine disrupting and in what quantities. The goal is to develop research methods where, for example, zebrafish can be used to investigate a chemical and at the same time get a reasonably accurate indication of how the same chemical will behave in humans.

‘The interesting thing is that large parts of our hormone system are evolutionarily conserved, so it's not that different from zebrafish to humans’, says Henrik Holbech.

The purpose of the research work is to identify biomarkers, for example gender change or the influence of some proteins and make the biomarkers standardised tests that everyone uses in identifying potential risk chemicals.

The standardised tests must subsequently lay the foundations for legislation in this area.

‘Right now, the rules are not perfect. For example, there are strict requirements for pesticides and biocides that need to be investigated for endocrine disrupting effects, but there are no requirements for industrial chemicals. At the same time, the EU has a rule that the industry, the manufacturer or the importer are always responsible for testing whether given chemicals meet the applicable standards. In the United States, things look different, and responsibility lies with the Environmental Protection Agency’, explains Henrik Holbech.

The EU has taken this issue seriously since 2018, but the best news is that we have been doing it in Denmark for a much longer time.

Nanosilver can be dangerous and it's everywhere

Let’s return to the nanoparticles. Plastic is far from the only material that can end up in nanosize in the environment and in us. More and more materials are being made in nanosizes because the properties of a material can change dramatically as it goes from large to really small. This means that some materials can have very attractive properties in nanosize, but it also means that something that is not dangerous to us humans in large format can be very dangerous when it comes in nanosize.

On the positive side, nanoparticles can be used for everything from wastewater purification and oil cleaning to transporting medicine around the body or knocking down infections. An example is nanosilver, which has been shown to have an antibacterial effect, which large-scale silver possesses only to a limited extent. Therefore, some manufacturers of band-aids line the inside of their products with nanosilver so that they can better prevent infections from occurring. Fridge manufacturers do the same on the inside of the refrigerators to prevent bacteria from forming small smelly colonies. The same goes for packaging that can make food last longer. Finally, some lotion manufacturers have found that adding silver nanoparticles to their products or in stockings prevent smelly feet.

On the negative side, researchers at Professor Frank Kjeldsen's research group at the Department of Biochemistry and Molecular Biology have found that nanosilver has an affinity for binding to the mitochondria inside our cells. The mitochondria are the small power plants of our cells, and it doesn't take much imagination to imagine that it is generally a bad idea that nano-sized extraneous particles disturb them in their work. Among other things, the group’s work has shown that if cells are affected by too many silver nanoparticles, cell death can be provoked. A contributing reason is that the silver nanoparticles create so-called free radicals that can bind to and destroy all possible compounds from proteins to fats inside the cells.

‘It's a double-edged sword, and I don’t want to only look on the dark side. The truth is, there are probably more scientists trying to develop new nanoparticles for the benefit of humanity than there are scientists trying to figure out how they can be harmful. We cannot stop the development of nanoparticles because they bring so much good with them, but we need to be much better at identifying those that are causing problems, and then we must have enough knowledge to be able to make sensible legislation. After all, in the case of silver nanoparticles, it must be enough to avoid using too many of them in different products and to tighten up the legislation. However, this does not apply to other nanoparticles that can be very beneficial without having an accompanying negative effect. We must try to make sustainable nanotechnology’, says Frank Kjeldsen.

Some of the world's most advanced equipment

In Frank Kjeldsen's laboratory, they have one of the most advanced tools for studying the effect of nanoparticles on cells. More specifically, we are talking about one of the world's most advanced mass spectrometers used to analyse proteins in cells. Basically, proteins express how the cell is doing, and if nanoparticles affect the expression of proteins and thus the well-being of the cell, scientists can measure it.

With the mass spectrometer, scientists can simultaneously analyse 60-70 percent of all proteins in a cell. They can see if there is more of some and less of other proteins. If, as in the example of the silver nanoparticles, the nanoparticles result in a lot of free radicals inside the cells, the cell will produce more proteins to manage the free radicals. The researchers will be able to see this, and thus they can determine that a given nano-sized substance results in the formation of harmful free radicals.

‘In our experiments, we found, among other things, that nanoparticles usually have the ability to enter the body and encapsulate themselves in proteins, so that the nanoparticles appear as a natural part of the body and not a foreign body. This type of experiment gives us a much better overview of the effects that nanoparticles can have on an organism or on us’, explains Frank Kjeldsen.

Combination effect can prove even more dangerous

His vision for the future is that legislation will be tightened up in this area, so there will be more control over the possible negative effects of new nanoparticles on us humans before they are released to the market and our surroundings.

However, the task is by no means straightforward. As an example, there are currently 3,000 nanoparticle products and they can interact differently with each of the body's 200 different types of cells. In effect, this means that every single type of nanoparticle needs to be tested against each of our cells to see if they can have a negative impact.

However, the task does not end there because there is another layer: the nanoparticles can potentially interact with each other, and then the negative effect of two different nanoparticles may be greater than the sum of the nanoparticles together.

‘We are facing a huge challenge because we need to find methods that can keep up with the development and test the vast numbers of nanoparticles already out there – which will only increase in the future’, says Frank Kjeldsen.

He is working, among other things, to get financial support to develop an improved protein platform, which will allow his laboratory to test 10 times as many nanoparticles on a daily basis.

‘That is where we stand today. The industry continues to spit out more and more nanoparticles to the market, and we need to find methods to give the authorities some concrete data to base their legislation on’, says Frank Kjeldsen.

Size matters

Like his colleagues, Professor Horst-Günter Rubahn, SDU NanoSYD, believes that the world faces a major task. One of the biggest problems is, not strangely, size.

Nanoparticles are defined as particles sized between 1 and 100 nanometers, and although this may sound small, there is actually a big difference between them. If the particles are very small, they can penetrate the cells and be reactive, but if they are only slightly larger, they do not have this effect.

An example is gold. Nanogold in one size can be very reactive and potentially harmful to cells, but if the particles are just a bit larger, it's just small chunks of gold.

‘If we get below 10-20 nanometers, many materials are potentially dangerous. Therefore, there is a major task in making the industry better at producing nanoparticles in sizes where their properties as nanoparticles are well preserved but where they don’t become dangerous. Currently, the industry just makes a lot of nanoparticles, and they span a wide range of sizes. They need to become better at narrowing the gap so that not so many of the harmful nanoparticles enter the environment. We usually say that to make nanoparticles, you have to do it properly. If you do it cheaply, you get a lot of the toxic nanoparticles’, says Horst-Günter Rubahn.

Methods for finding nanoparticles

He works closely with Frank Kjeldsen and says that one of the big questions right now is whether nanoparticles in the small quantities that we are exposed to have any effect on our health at all?

As it currently appears, according to Horst-Günter Rubahn, the risk seems minimal, but this can change rapidly as nanoparticles enter more and more of our surroundings. In addition, no one currently has an overview of the possible consequences in the long run.

‘As an example, cancer evolves over several decades, and it is not currently possible to say whether nanoparticles increase the risk of developing cancer. That is why the EU is also very keen to regulate the area’, says Horst-Günter Rubahn.

To help politicians make the best possible decisions, the researchers at the Horst-Günter Rubahn Institute are developing various methods to quickly identify if a nanoparticle is harmful. One method is the one that Frank Kjeldsen is working on, but other methods are also at play. Among other things, the researchers are working on different methods to identify, for example, whether a blood sample or a water sample contains nanoparticles that should not be there.

One method involves nanoparticles in a liquid in small tubes. The researchers shoot a laser at the nanoparticles, and depending on the light scattering, they can determine the size and number of nanoparticles. Previously, the researchers have run the liquid through a series of small tubes that divide the particles into sizes so that in one sample there are only nanoparticles between, for example, 20 and 50 nanometers, while in the next sample there are only nanoparticles between 50 and 100 nanometers.

‘This way we can isolate the particles that we want to see. For example, if we have identified that particles above 80 nanometers are not harmful, we can leave them out and then concentrate on identifying the smaller ones’, explains Horst-Günter Rubahn.

Another method involves small ceramic plates with small recesses. The recesses have different sizes that can collect nanoparticles according to their size. The ceramic plates resemble small baking molds for muffins.

‘We are developing various tests that we hope to have ready within the next two years. Then it is up to the authorities to decide whether it should be standard for the industry to test the size of the particles in their products to see if any of them are down to the size where they can become harmful. We also try to develop a similar test for consumers, so that, for example, companies can test the nanoparticle size in the products they buy from others. If a cosmetic manufacturer uses nanoparticles in their lotions, they would like to be able to tell consumers that the size of the nanoparticles makes them effective, but not dangerous, and then it is best to have the tests done yourself’, says Horst-Günter Rubahn.

Nanoparticles may kill cancer

Horst-Günter Rubahn states that nanoparticles cannot be avoided in either the present or the future, and that it is all about understanding what they can do for us or, at worst, do to us. Then we have to legislate and regulate so that we get as many benefits as possible from the nanoparticles at the lowest possible cost to our environment and our health.

Nanoparticles have the potential to revolutionise many industries, including the healthcare system. For example, according to Horst-Günter Rubahn, nanoparticles can be developed that have the property of binding to cancer cells. In this case, the nanoparticles can be passed into the body and subsequently heated up to kill the cancer cells.

‘There will be many opportunities to use nanoparticles like that, but unfortunately cancer is a very complex disease. In this area, we still lack knowledge and research to finally reach our goals’, says Horst-Günter Rubahn.

Drawings: Mikkel Larris, SDU