Isotopic fingerprints of environmental contaminants | UDaily

Plants need phosphorus, an essential nutrient, to grow. This nutrient is found in fertilizers and applied to crops to increase yields.

One downside to excess phosphorus in this agricultural environment is that it can leach into lakes, rivers, and other bodies of water, causing water quality problems such as algae blooms, which rob the water of oxygen and create dead zones, killing fish and other aquatic life.

Phosphorus in the environment occurs in two forms: organic and inorganic. Inorganic phosphorus is the simplest form and is most easily accessible to soil microorganisms and plants. Organic phosphorus compounds are much more complex, making them difficult to identify and track in the environment.

Scientists often use isotopes of an element as a way to track its presence in the environment. This is because isotopes of an element have the same number of protons, but different numbers of neutrons. They are essentially families of the same element with slightly different masses. For example, carbon-13 is an isotope of carbon that has been useful in determining the age of groundwater, while carbon-14 has helped researchers date material artifacts, such as ceramics, to a particular time period.

Scientists have long developed methods to measure the oxygen isotopes of inorganic phosphates in the environment. But there is no such method to measure the isotopes of organic phosphates.

“The current method requires removing the phosphate from a molecule,” said Deb Jaisi, a professor of environmental biogeochemistry at the University of Delaware in the Department of Plant and Soil Sciences. “Once it’s removed (hydrolyzed), it’s no longer organic and can no longer be used for source tracing because that change alters the original isotopes of the molecule.”

That’s where Jaisi and Tony Hollenback, a recent Ph.D. graduate from Jaisi’s lab, come in.

The duo has published new research on Journal of the American Society for Mass Spectrometry which details the development of a new method for measuring the isotope fingerprints of organic phosphate molecules using mass spectrometry techniques. The method uses an instrument called the Orbitrap electrospray ionization-based isotope ratio mass spectrometer (Orbitrap IRMS), an advanced instrument designed for such analyses. UD hosts one of only nine such Orbitrap IRMS instruments nationwide.

Tracing the origin of pollutants

Studying the origins of environmental contamination is necessary if researchers hope to find solutions to eradicate contamination, identify best management practices, and/or remediate a contaminated site.

In the Chesapeake Bay, the watershed from which Hollenback and Jaisi took soil samples, phosphorus is a major pollutant that affects the health of the bay. The state of Maryland has a plan for how to reduce the bay’s phosphorus by a certain amount by 2025.

Phosphorus can degrade water quality by triggering algal blooms. Algal blooms suck oxygen from the water, killing fish, plants, and other organisms.

“Simply put, we want to know where the phosphorus is coming from,” Jaisi said.

Jaisi and Hollenback looked at one molecule, specifically phytate. All the plant seeds that are fed to pigs and chickens, on the Delmarva Peninsula or anywhere else, are very high in phytate. Hollenback explained that pigs and chickens are “non-ruminants,” meaning they don’t have certain enzymes to break down phytate. That means phytates get concentrated in the animals’ manure.

“If that manure is applied to crop fields as fertilizer, the phytate becomes concentrated in the soil,” Hollenback said. “During rainfall events, that phytate is then transferred to nearby streams, which then flow into the bay. Now there are microbes in the water, whether it’s fungi or bacteria, and they have enzymes to break it down.”

When fungi or bacteria break down phytate, they release inorganic phosphate, the most bioavailable form of phosphorus, into the water, thus allowing the growth of harmful algae.

“So we need to be able to monitor it,” Hollenback said, “because it is potentially a very important piece of the puzzle in trying to solve this problem.”

A new method

To do this, Hollenback and Jaisi developed a method to measure organic phosphate isotopes using the Orbitrap IRMS, which they acquired with funding from the U.S. Department of Agriculture’s Equipment Grant Program. The experiment was conducted in UD’s Patrick T. Harker Interdisciplinary Science and Engineering Laboratory.

The UD researchers collected soil from a farm outside Crisfield, Maryland, near East Creek, a tributary of the Chesapeake Bay. This particular soil, which had long been fertilized with animal manure, was rich in phytate, the organic phosphorus compound most commonly found in farmland.

“The goal of this research was twofold,” Hollenback said. “One [goal] was to look at the biology of the soil, to see what interacts with the phytate. The other was to explore and track the oxygen isotopes in the phytate using the IRMS Orbitrap.”

The researchers incubated the soil samples in lab conditions for about three months. They also “spiked the soil” with extra phytate, Hollenback said. This helped them see if any changes occurred in the soil. They even added isotopically labeled water to study how the phytate would circulate through the system.

“Many of the changes we saw with the extra phytate formed were biological,” Hollenback said. “A key finding of this research was that the phytate molecule faithfully retains its isotopic fingerprints.”

The Orbitrap MS method originated at the California Institute for Technology, but that institute used it for other compounds, not phosphate. UD researchers inject a solution containing purified soil phytate into the instrument using a high-precision syringe. The molecules pass through a mass filter that removes any contaminating ions. The mass is then measured with extreme precision in the Orbitrap mass analyzer by tracking a band of ions as it rotates around a central spindle.

The aptly named Orbitrap MS “traps” ions to measure molecules, both composition and isotopes. Since this measurement was made for isotopes, a new name was coined Orbitrap IRMS (Orbitrap isotope ratio mass spectrometry).

“The phosphate isotope in the phytate molecule has not been touched by any process,” Jaisi said. “We found that the isotope signature of the molecule remains the same.”

This is a breakthrough. If the isotope of a molecule stays the same, it satisfies the first requirement for identifying the source of a molecule or contaminant in the environment. If the isotope of a molecule were to change in any way, it would essentially erase the origin of the molecule.

“We now have a new wheel,” Jaisi said. “A new wheel that we’ve developed in terms of tools. And in terms of isotopes, because the molecule has a signature that stays the same, the discovery can be used for source tracing for this compound and can be used similarly for other molecules.”

The next step is to see how it works in the real world. The good news is that field testing is underway.

Additionally, the method Jaisi and Hollenback developed with the Orbitrap IRMS can be used across a variety of disciplines and chemical compounds. It is a great asset for UD, as other research teams at the University are already using the Orbitrap IRMS to study various compounds. These include explosive compounds and even “forever chemicals” known as PFAS, perfluoroalkyl and polyfluoroalkyl substances.

“It’s such a powerful tool,” Jaisi said. “While it’s not very routine for anyone to be able to use it without specific training, the methods are continuing to be refined with the tool. It’s very early in its development, but it’s a great opportunity for anyone to jump in and develop the science of leadership using the tool.”

The paper, Position-Specific Oxygen Isotope Analysis in Inositol Phosphates by Using Electrospray Ionization-Quadrupole-Orbitrap Mass Spectrometry, was written by Anthony J. Hollenback and Deb P. Jaisi. It was published in the Journal of the American Society for Mass Spectrometry. Funding was from the National Science Foundation and the U.S. Department of Agriculture.