Unveiling EPA 1634: Pioneering 6PPD-Quinone Analysis and its Environmental Implications

After the landmark publication in Science led by the University of Washington, the specialty team at Eurofins was eager to see how our capabilities could benefit this issue. We began our research and crafted a series of 6PPD-quinone webinars that took clients by storm. These webinars were the first of their kind that addressed 6PPD-quinone and theorized how to move forward. Shortly afterward, EPA took notice of our work and reached out in an effort to address the issues discussed. In January 2024, the EPA published the single-lab validation for 6PPD-quinone.

The main issue being that juvenile Coho Salmon (Oncorhynchus kisutch) exposed to a single compound, 6PPD-quinone, was responsible for urban runoff mortality syndrome (URMS). This mortality occurred at relatively low concentrations with an LC50 estimate at around 100ng/L (ppt). Excellent detective work would be needed as 6PPD-quinone was not an industrial component, but rather a product of the reaction between the parent compound 6PPD and naturally occurring ozone.

Since the groundbreaking publication, other manuscripts have begun to indicate that rainbow trout (Oncorhynchus mykiss) are also susceptible to 6PPD-quinone. Although thankfully not as acutely as Coho salmon, Rainbow trout are found throughout the world, making this a global issue.

Coho salmon are both socially and economically significant. Monitoring the contaminants we consume is vital to long-term health and increasing effectiveness of potential treatments, but more importantly, if the salmon numbers dwindle there is no viable fishery. This would yield negative impacts to the environment and the native tribes that depend on the Coho, both culturally and for sustenance.

In addition to the impact on select fish, 6PPD-quinone has also been detected in humans. Human toxicology has yet to be established, but the early research indicates a concentrative effect on pregnant women. With each study, it became clear that characterizing aqueous concentrations of 6PPD-quinone is key to delineating this emerging contaminant.

That’s where our specialty team came in. Up until this point, a commercially available analysis didn’t exist for 6PPD-quinone. Our team is the first commercial lab to respond with a robust quantitative method for 6PPD-quinone once the emerging contaminant had been identified, making our development of this method the first of it’s kind.

Technical Challenges

When 6PPD-quinone was discovered in late 2020, there was no analytical standard available. The only similar standard was the parent compound 6PPD. By using advanced Time-of-Flight (ToF) instrumentation (that doesn’t need analytical standards), the Eurofins team was able to isolate 6PPD-quinone from local roadways. The advanced TOF instrumentation is not a typical analysis platform, but the Eurofins network maintains five such instruments. It was through this technique that Eurofins was able make key decisions without an analytical standard.

Development posed a few technical challenges including the difficulty of the matrix. Confident quantitation of 6PPD-quinone in stormwater is critical in assessing 6PPD-Q concentrations. These concentrations can change quickly and the associated change in matrix cannot impact analysis. This dynamic soup is comprised of oils, salts, surfactants and other automotive and industrial chemicals. All of this is in addition to comingled biological material and an array of particulates.

Once we tackled that technical challenge, we dived into navigating isotopes of 6PPD-quinone with analytical standard vendors. Without isotopes, precise quantitation would not be possible, especially in complex matrices. As of January 2024, there were only three isotopes of 6PPD-quinone on the market.

The role of the environmental analytical laboratory is always anchored by neutrality. Both the regulators and the regulated have to be confident in the data produced. In this case, a wide swath of stakeholders are involved. Federal agencies like the EPA and the Department of Transportation (DOT), as well as State and Tribal governments require answers to the contaminant. This is contrasted by chemical producers and includes tire manufactures that must comply with federal guidelines surrounding tire safety additives. All sides, big and small, need confidence in their data.

The Method Overview

The overview of this new 1600 series method is relatively straightforward. Once the aqueous sample is collected in the 250mL amber glass bottle, it undergoes overnight shipping to the lab on wet or blue ice. Results are typically delivered around three weeks later.

During this time, the volume is recorded before the sample is spiked with an isotope of 6PPD-quinone. It’s gently shaken and allowed to equilibrate before the sample is extracted and cleaned up using solid phase extraction (SPE). This step serves a dual purpose; it eliminates bulk interferences and naturally concentrates the sample.

A final isotopically labeled standard is added just before injection to aid in determine the efficiency and success of the SPE. The sample is then injected into the liquid chromatograph (LC) and the masses for 6PPD-quinone while the isotopes are monitored by the tandem mass spectrometer (MS/MS). Lastly, data are processed against an established calibration using the gold-standard of quantitation, isotope dilution quantitation.

What the Future of the Method Holds

The single-lab validation was published in January 2024 so a multi-lab study should begin shortly. Despite this, clients can begin requesting EPA 1634 as soon as today. We expect more and more labs will begin to align with the new method during the multi-lab process. For additional information, readers can visit our website. They can also find the official press release here: EPA 1634.