Molecular sleuthing using mass spectrometry to protect Pacific salmon populations

Jul 14, 2021 | Blogs, Environmental / Industrial | 0 comments

Read time: 5 minutes

When Pacific salmon start to die in large numbers, with no identified cause, people take notice. This concern stems not only from the possibility of an environmental issue that could additionally affect human health, but also, in this case, from the cultural and economic importance of Pacific salmon.

The problem

It turns out the unexplained die-off of salmon isn’t a new problem. A study by Zhenyu Tian et al. found that urban runoff mortality syndrome (URMS) has been documented in US Pacific Northwest coho salmon for decades.1 In fact, they cite work suggesting that 40%–90% of salmon may die when returning to freshwater spawning sites that are impacted by urban runoff. Despite the long history of URMS, however, an empirically backed, plausible cause for these die-off events had not been identified until recently.

The (exhaustive) investigation

Freshwater sites impacted by urban runoff are likely to contain a variety of anthropogenic chemicals that can affect the health of plants and animals in that ecosystem. Previous research indicates that water collected from sites where salmon die-offs have occurred has a chemical composition similar to that observed for roadway runoff and tire extracts. Using this finding as a jumping-off point, Tian et al. focused on orthogonal analytical and semi-preparative separation chemistries combined with toxicity studies to reduce the number of unknown compounds detected by mass spectrometry from >2,000 molecular features down to 7 potential toxicants.

By using the consistency of the toxicological response in fractions collected from the different column phases, the authors were able to efficiently follow the suspect toxicant through the purification process. Using the power of accurate mass along with fragmentation analysis, an empirical formula for the dominant chromatographic peak in the final toxic fraction was determined. A subsequent literature review suggested the anti-ozonant 6-PPD [N-(1,3dimethylbutyl)-N’-phenyl-p-phenylenediamine, which is present at percent levels in many tire rubber formulations, as a strong candidate for the parent compound of the suspected toxicant. Mass spectral comparison with authentic standards of 6-PPD and ozonated 6-PPD, as well as NMR of the purified toxicant, confirmed 6-PPD quinone (an oxidation product following from reaction with ozone) as the causal agent in the URMS-related salmon die-offs investigated by Tian et al.

So, what next?

The findings in this study raise important questions about regulatory issues that could arise around 6-PPD and similar anti-ozonants in tire rubber as they relate to salmonid conservation. It also seems unlikely that toxic effects would be limited to salmonids, which leads to the broader question of how 6-PPD quinone might affect other freshwater species. And lastly, it’s important to note that 6-PPD is added to tire rubber as a sacrificial chemical component, which means that it will generate a variety of degradation products that must be considered.

In light of the potential environmental burden of 6-PPD (up to 500 g/passenger car or 10 kg/heavy truck1) and related anti-ozonants in tire rubber, it is probable that other degradation products exist. The likelihood of structural homology between these products and 6-PPD quinone could warrant investigation into their toxicological effects as well. At present, SCIEX, Phenomenex and Eurofins are interrogating water samples known to contain 6-PPD quinone for structurally related 6-PPD degradation products using the SCIEX X500R QTOF System.


The work of Tian et al. underscores the power of mass spectrometry, particularly accurate mass analysis, when it comes to investigating unknown environmental contaminants. It is also an example of the gains that can be made when experts from a wide swath of scientific disciplines come together to understand a complex environmental challenge. Let’s keep collaborating as a scientific community (yes, with vendors too!) so that meaningful work like this study can continue to advance our understanding of our impact on the environment.

Check out this on-demand webinar about 6-PPD testing to learn how Eurofins, SCIEX and Phenomenex are working together to understand testing methods and investigate the possible presence of structurally related 6-PPD degradants.


1 Zhenyu Tian, Haoqi Zhao, Katherine T. Peter, Melissa Gonzalez, Jill Wetzel, Christopher Wu, Ximin Hu, Jasmine Prat, Emma Mudrock, Rachel Hettinger, Allan E. Cortina, Rajshree Ghosh Biswas, Flávio Vinicius Crizóstomo Kock, Ronald Soong, Amy Jenne, Bowen Du, Fan Hou, Huan He, Rachel Lundeen, Alicia Gilbreath, Rebecca Sutton, Nathaniel L. Scholz, Jay W. Davis, Michael C. Dodd, Andre Simpson, Jenifer K. McIntyre, Edward P. Kolodziej. A ubiquitous tire rubber–derived chemical induces acute mortality in coho salmon. Science. 2020, 371 (6525), 185-189. DOI: 10.1126/science.abd6951



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Matt is the Market Development Manager for the Cannabis, Food and Environmental markets in the Americas at SCIEX. Matt brings 15 years of LC-MS experience to this role, spanning the pharmaceutical, environmental, food and cannabis industries. Matt loves talking about all things mass spectrometry, so reach out if you have any questions –



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