Toxicity tests are currently still carried out mainly on livestock. Within the context of environmental risk assessment, this is done most commonly on fish. An alternative method developed by Eawag researchers is currently overcoming crucial hurdles to its widespread use in practice. Thanks in part to the persuasive efforts of the scientists involved.

Professor Kristin Schirmer (right) and her colleague Melanie Fischer. Foto: Basil Stücheli

How do you measure whether a chemical substance is safe for the environment? By exposing live fish to increasing doses of this chemical until they finally die. And how do you measure whether the water from wastewater treatment plants is really clean? By keeping fish as living sensors. They indicate if there are problems with the water quality, as an early warning system, so to speak.

Professor Kristin Schirmer, Head of the Environmental Toxicology Department at Eawag, had long come to realise that there would have to be another way of doing this. She calls the current standard procedure for measuring water quality an “outmoded, crude test”. Nevertheless, the so-called acute fish toxicity test is one of the most widely used tests in environmental regulation. Schirmer is convinced that it is time to change this. So how do you make sure that any danger is detected without experimenting on living organisms?

Schirmer and her team have established a method involving a cell line from the gills of rainbow trout. They proceeded on the assumption that acute fish toxicity mainly affects the gill cells in the fish. The method that has since been established is something like a standardised fish substitute that can be used in different laboratories and should provide the same results everywhere. The breakthrough came with a round-robin test in various laboratories around the globe. The scientific article on this was published in the respected journal “Toxicological Science” in April 2019. Schirmer and her team have demonstrated that the methodology, which has long been established in their own laboratory, works reliably and can also be reproduced in other laboratories.

Gill cells can be tested for vitality using fluorescent dyes
Photo: Basil Stücheli

The gill cells can be tested for vitality using fluorescent dyes. In doing so, they react to a whole spectrum of chemical substances in a similar way as living fish. “This has confirmed our assumption that toxins initially affect the gills,” said Schirmer. There were a few outliers, particularly with some neurotoxic substances. Eawag researchers are, therefore, planning to extend the procedure to include nerve cells. They are already conducting intensive studies on the effects of chemicals using cell lines from the liver and intestine of rainbow trout. They are also experimenting with the cultivation of cells on chips, on which cell vitality can be measured in real time on the basis of the electrical resistance provided by the cells. These chips should also make remotecontrolled, fully automatic measurements possible. “Our vision is to fully simulate a living fish by combining different cell types,” said Schirmer. Her team is already working on the possibility of using computer programs to investigate the effects of chemicals in the future.

“Our vision is to fully simulate a living fish by combining different cell types,” said professor Kristin Schirmer, Head of the Environmental Toxicology Department at Eawag

As ruthless as the current test may appear, at least it measures what it is supposed to measure. Or does it? “It's an illusion to assume that every test with live fish yields clear results,” says Schirmer. Because the test is primitive both in terms of its concept and its validation. In terms of comparability and reliability, the new test is much more stringent, and the requirements placed on laboratories are more rigorous. This has persuaded specialists to get behind it. It has already attracted a great deal of interest from industry. The round-robin test was co-financed by the European Chemical Association. After all, the revolution would not only mean fewer animal experiments, but also simpler, cheaper and more standardised methodology. Specific test requests soon followed from industry, and Schirmer, together with her laboratory assistant Melanie Fischer and Eawag postdoctoral student Stephan Fischer, founded the Eawag spin-off aQuaTox-Solutions back in 2016.

The matter was “actually concluded from a research perspective” with the publication of the round-robin test. Nevertheless, Schirmer and her team will continue to invest a great deal of time in the project to ensure that the methodology is actually applied in practice. She is extremely motivated by the prospect of bringing something so important to society. Thus, she made regular visits to certification authorities and learnt how to convince not only her peers from the research community and technical experts on various commissions. The combined research and lobbying efforts ultimately led to the first toxicity test using cultured gill cells being ISO-certified in 2019. This earnt the researchers the 3Rs Award 2019 by the 3R Competence Centre Switzerland (3RCC). And Schirmer is confident that further progress will now be made quickly. She is currently working with experts on the certification of the gill cell test by the OECD.