Organic phosphate isotope method offers new way to track pollutants in the environment

Plants need phosphorus, an essential nutrient, to grow. This nutrient is found in fertilizers and applied to crops to increase their yields. One of the downsides of excess phosphorus in this agricultural environment is that it can leach into lakes, rivers, and other bodies of water, causing water quality issues such as algae blooms, which remove oxygen from the water and create dead zones, killing fish and other aquatic life.

Phosphorus occurs in the environment in two forms: organic and inorganic. Inorganic phosphorus is the simplest form and is most readily available 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 to track its presence in the environment. This is because isotopes of an element have the same number of protons, but a different number of neutrons.

These are actually families of the same element with slightly different masses. For example, carbon-13 is an isotope of carbon that has helped determine the age of groundwater, while carbon-14 has helped researchers date material objects, such as pottery, to a particular period.

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

“The existing method requires removing the phosphate from a molecule,” said Deb Jaisi, professor of environmental biogeochemistry in the Department of Plant and Soil Sciences at the University of Delaware.

“Once you remove it (hydrolyze it), it is no longer organic and can no longer be used for source tracking because that change alters the original isotopes of the molecule.”

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

The duo published new research in the Journal of the American Society for Mass Spectrometry detailing the development of a new method for measuring isotopic fingerprints of organic phosphate molecules using mass spectrometry techniques.

This method relies on a tool called the Orbitrap electrospray ionization-based isotope ratio mass spectrometer (Orbitrap IRMS), an advanced instrument designed for such analyses. UD is home to one of nine such Orbitrap IRMS instruments nationwide.

Monitoring the origin of pollutants

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

In the Chesapeake Bay, the watershed where Hollenback and Jaisi took soil samples, phosphorus is a major pollutant affecting the health of the bay. The state of Maryland has a plan to reduce phosphorus in the bay by a certain amount by 2025.

Phosphorus can degrade water quality by triggering algae blooms, which absorb oxygen from the water, killing fish, plants and other organisms.

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

Jaisi and Hollenback studied one molecule in particular: phytate. All plant seeds fed to pigs and chickens, on the Delmarva Peninsula or elsewhere, are very high in phytate. Hollenback explained that pigs and chickens are not ruminants, which means they don’t have certain enzymes to break down phytate. That means phytate becomes concentrated in the animals’ manure.

“If that manure is used as fertilizer for crops, the phytate gets concentrated in the soil,” Hollenback says. “When we have rain 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, allowing harmful algae to grow.

“So we need to be able to track it,” Hollenback said, “because it’s potentially a very important puzzle piece in trying to solve this problem.”

A new method

To do this, Hollenback and Jaisi developed a method for measuring organic phosphate isotopes using the IRMS Orbitrap. The experiment was conducted at UD’s Patrick T. Harker Interdisciplinary Science and Engineering Laboratory.

The UD researchers sampled soil from a farm outside Crisfield, Maryland, near East Creek, a tributary of the Chesapeake Bay. This particular soil, which has been fertilized with animal manure for a long time, is high in phytate, the organic phosphorus compound most commonly found in agricultural soils.

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

The researchers incubated the soil samples under laboratory conditions for about three months. They also “spiked” the soil with additional phytate, Hollenback said. This allowed them to see if any changes were occurring in the soil. They even added isotope-labeled water to study phytate cycling in the system.

“A lot of the changes we saw in the formation of additional phytate were biological in origin,” Hollenback said. “One of the key findings of this research is that the phytate molecule faithfully retains its isotopic fingerprints.”

The method using the Orbitrap MS was invented by the California Institute for Technology, but that institution has used it for other compounds, not phosphate. The UD researchers inject a solution containing phytate purified from soils into the instrument using a high-precision syringe.

The molecules pass through a mass filter that removes all contaminating ions. The mass is then measured very precisely in the Orbitrap mass analyzer by following a band of ions as it rotates around a central axis.

The aptly named Orbitrap mass spectrometer “traps” ions to measure molecules, both their composition and their isotopes. Since this measurement was done for isotopes, a new name was coined: Orbitrap IRMS (Orbitrap Isotope Ratio Mass Spectrometry).

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

This is a major breakthrough. If the isotope of a molecule remains the same, it meets 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.

“Now we have a new wheel,” Jaisi said. “We have developed a new wheel at the instrument level. And at the isotope level, since the molecule has a signature that remains the same, the discovery can be used for tracking the source of that compound and can be used analogously for other molecules.”

The next phase is to see how it works in a real environment. The good news is that field testing is underway.

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

“It’s a very powerful instrument,” Jaisi said. “While it’s not common for anyone to be able to use it without specific training, the methods continue to be optimized with the instrument. It’s still in its early stages, but it’s a great opportunity for anyone to jump in and develop the science of leadership using this instrument.”