Nitrate stable isotope analysis

Nitrate stable isotope analysis: the need for a new method. By Dr Calum Preece

Stable isotope analysis of nitrate could make many valuable contributions to environmental research, but there are several disadvantages to existing methodologies that preclude this. As the problem of nitrogen pollution becomes more urgent, and environmental stakeholders begin to recognise that a growing volume of nitrates in our groundwater systems could pose a significant risk to the health of people and ecosystems, it is vital that the scientific community adopts a faster, more cost-effective method.

Thankfully, such a method was recently developed by Dr Mark Altabet and Dr Len Wassenaar. It removes many of the barriers to entry that previous methods imposed, meaning that many smaller labs can now viably carry out nitrate isotope analysis, without any expensive investments or significant changes to workflows. Here, we will explore the role nitrate isotope analysis could play in fighting pollution and protecting the environment, detail the existing methods and their disadvantages, and explain how the new method could overcome these challenges.

What are the risks of environmental nitrogen, and how does stable isotope analysis help?

As a nutrient, nitrogen is consumed during the natural processes of many plants and bacteria; however, when too much enters our water systems, it can cause serious problems. In high enough quantities, nitrate can poison sources of drinking water. Excess nitrate in rivers, streams and lakes can lead to eutrophication, resulting in algal blooms - which can be toxic - and anoxia, which reduces the available oxygen within a body of water and can kill fish at alarming rates.

It is simple to measure concentrations of nitrogen compounds and quantify the problem, but stable isotope analysis could play a different role. Wassenaar comments: “The isotopes of nitrate can tell us about the sources and the processes that will help to remediate nitrate pollution.”

Two primary sources of environmental nitrates are agriculture and wastewater treatment. With stable isotope analysis, researchers can detect the mismanagement of fertilisers and identify wastewater treatment processes that are ineffective at removing nitrates from water before it is released into the environment.

Unfortunately, this valuable analysis is cost-prohibitive, due to challenges with the most common methods. These methods produce reliable results, but are also slow and difficult to reproduce at scale. Until now, this has acted as a barrier to the wider use of stable isotope analysis.

What are the challenges with the existing methods?

There are two existing methods for nitrate stable isotope analysis. The first is called Cd-azide reduction method, and uses cadmium and azide to react with nitrate and convert the liquid into a nitrous oxide gas, which can then be analysed. Wassenaar says this method “works very well,” but also notes: “The problem is that both cadmium and azide are very toxic. It’s difficult to get a lab licence to use it. You have to treat it as an extreme toxin and have all of the associated safety protocols in place. It also produces a toxic waste, which you then have to dispose of.” Based on these factors alone, laboratories that use this method are rare.

The other method is referred to as the bacterial denitrifier method. It uses strains of bacteria to convert nitrate to nitrous oxide gas for analysis. Bacteria must be stored anaerobically, and sample preparation must be carried out under the same conditions. This method also requires distinct and uncommon skill sets: microbial ecology, for the cultivation and maintenance of the necessary bacterial cultures; and stable isotope mass spectrometry to carry out the analysis.

The bacterial denitrifier method is more common than Cd-azide reduction method, but both methods place high demand on laboratories. Consequently, there are relatively few labs in the world that provide stable isotope analysis of nitrate using either method. Wassenaar says: “In my career over the past 10 years, it has been very difficult to process samples. There were so few labs, so the backlog could be six months to a year, and the price was high.” These, he said, became significant barriers to the use of nitrogen stable isotope analysis.

What is the new method for the stable isotope analysis of nitrate?

In 2019, Altabet and Wassenaar published a new method of stable isotope analysis that overcomes the downsides shared by the previous methods. Wassenaar explains: “One of our goals was to develop new methods that could be readily adopted by laboratories worldwide, and this is where the titanium technique came from.”

The new method uses a titanium (III) chloride solution, which reacts with the nitrate in a sample to produce nitrous oxide gas. Unlike previous methods, samples can be prepared in fewer than 24 hours. Wassenaar states: “It’s easy. It can be done in any laboratory; it doesn’t require costly fume hoods or produce toxic waste. You don’t need to cultivate bacteria. The chemicals are readily available and cheap - it’s almost a perfect technique.”

Without the safety concerns or practical challenges associated with the earlier methods, it is fast and simple for laboratories to adopt this new technique. With a mass spectrometry instrument optimised to analyse stable nitrate isotopes using this method, labs can further accelerate workflows and make analysis more efficient.

Wassenaar insists that the method is almost perfect, but not quite; it only converts around 80% of the nitrate in a sample to nitrous oxide, unlike the 100% conversion achieved with the Cd-azide reduction and bacterial denitrifier methods. Additionally, since the titanium method is so new, it has not been used on the same number of sample types as the two more established methods; as such, there is still work to be done to refine this new method and deliver ever-improving results.

There is still a place for the Cd-azide reduction and bacterial denitrification methods, especially for those laboratories that are already providing this type of analysis. However, the new method could provide greater access to these insights worldwide.

Using data from stable isotope analysis, stakeholders can take action against polluters, develop more effective wastewater treatment processes, and begin to address the role of agriculture in nitrogen pollution. Using this method, we hope that small labs can make nitrogen a much larger part of the conversation about pollution in the environment.

Dr Calum Preece is Environment Product Manager at Elementar UK

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