Exploring the Environmental Footprint of MS/MS Techniques to Determine Endocrine-Disrupting Chemicals

In a joint study between the University of Duisburg-Essen (Essen, Germany), the Institute for Environment and Energy, Technology and Analytics (IUTA e. V., Duisburg, Germany) and the cooperation laboratory (KL) of Ruhrverband and Emschergenossenschaft/Lippeverband (EGLV) (Essen, Germany), the green analytical chemistry tool Analytical GREEnness (AGREE) and the white analytical chemistry (WAC) framework were used to conduct a comparative analysis of gas chromatography-tandem mass spectrometry (GC–MS/MS), liquid chromatography-tandem mass spectrometry (LC–MS/MS), and the yeast-cell-based reporter gene assay (A-YES). Their assessment critically evaluated each method's environmental impact, efficiency and practicality. An overview of potential improvements inspired by their work offers a path to the further enhancement of not only the greenness of the methods, but also their feasibility. Through the incorporation of these advances, the researchers are confident that each analysis can increase its scores in AGREE and WAC. LCGC International spoke to Michelle Klein from the Faculty of Chemistry of the University of Duisburg-Essen (Essen, Germany) about this analysis, and the recent paper that resulted from the work.

Your paper (1) discusses the use of the green analytical chemistry tool Analytical GREEnness (AGREE) and the white analytical chemistry (WAC) framework to conduct a comparative analysis of gas chromatography-tandem mass spectrometry (GC–MS/MS), liquid chromatography-tandem mass spectrometry (LC–MS/MS), and the yeast-cell-based reporter gene assay (A-YES) to determine the presence of endocrine-disrupting chemicals in aquatic environments. What sort of negative effects are associated with these endocrine-disrupting chemicals?

Exposure to endocrine-disrupting chemicals (EDCs) has been closely linked to adverse effects on both environmental and human health. The endocrine system, which regulates vital physiological functions such as development, metabolism and reproduction, is particularly vulnerable to these interferences. Even at trace concentrations of < ng/L, certain EDCs like estrogens can disrupt the hormone signaling system. These chemicals include a wide range of substances, from natural and synthetic hormones (e.g. androgens, estrogens, progestogens) to pharmaceuticals, personal care products, pesticides, detergents and industrial compounds.

In freshwater environments, aquatic organisms such as fish and amphibians are the first to be affected. EDCs can cause reproductive abnormalities, including feminization and reduced reproductive success due to altered sex ratios or infertility. They can also lead to behavioral changes, such as disrupted mating patterns or developmental abnormalities. Over time, these impacts can result in population declines, ultimately threatening ecosystem balance and stability.

Are there any chemicals that are more harmful than others? How is their environmental impact determined?

Organic pollutants, such as pharmaceuticals or plant protection products, are particularly concerning because they are specifically designed to have bioactive effects. The impact of EDCs can vary depending on factors like concentration and exposure duration. Some compounds are more potent than others, leading to harmful effects even at low concentrations. To determine their environmental impact, we rely on a combination of methods: chemical analysis to detect their presence, effect-based approaches to understand biological responses and ecological studies to assess their impact on organisms and ecosystems over time. This multi-faceted approach is essential to gain a comprehensive understanding of their effects.

In our environment, it is important to recognize the complexity of chemical mixtures. The challenge lies in the fact that the number of new or yet unknown chemicals in our surroundings is far greater than our ability to assess and monitor them effectively. Mixture toxicity and the dynamic interactions between these chemicals can significantly impact mechanistic studies, making it crucial to consider these combined effects when evaluating environmental risks. For a long time, traditional risk assessment models focused on individual substances in isolation, which does not assess real-world environmental exposure. Effect-based methods offer a powerful approach, helping us better understand the mechanisms, efficacy and toxicological impacts of EDCs. This underscores the need for more comprehensive and adaptive risk assessment frameworks that can account for these interactions and the complexities of environmental exposure.

Are techniques already in existence that can identify, locate or analyze certain reagent chemicals that are considered not environmentally friendly?

In the metric tools we used, such as AGREE, there are pre-set options for evaluating reagent chemicals. For example, you can enter CAS numbers or use the type and number of associated pictograms to assess their environmental impact. The WAC framework considers factors like genetically modified organisms, where a simple "yes" answer influences the results. However, in terms of finding more environmentally friendly alternatives, our search was based on literature research. These tools help us to evaluate current chemicals but finding alternatives still relies heavily on research and development.

Outside of the environmental benefits to your technique, are there any other advantages?

Outside of the environmental benefits, I believe there are several other advantages, depending on the number and types of metric tools used for method evaluation. For example, with the WAC framework, not only environmental impacts but also performance and practicability were considered. There are numerous other tools available, each with specific advantages, and by combining them, you should get different viewpoints of your method. The key is selecting the right combination to ensure a balanced and comprehensive evaluation of the method from multiple perspectives according to your ultimate analysis goals.

Briefly summarize your findings that you discuss in your article, and the conclusions you came to after reviewing these findings.

Our findings emphasize the importance of combining metric tools for a more thorough greenness assessment, as each has distinct strengths - AGREE focuses on environmental burden and safety, while WAC incorporates principles like performance and efficiency. In comparing methods for EDC analysis, including GC–MS/MS, LC–MS/MS and A-YES using tools like AGREE and WAC, we assessed their environmental impact, efficiency and practicality. Interestingly, no significant differences were observed between in-vitro assays like A-YES, YES, and ER-CALUX, though procedural variations exist. We also identified key areas for improvement, such as miniaturization, automation and in-situ SPE, which could enhance both greenness and efficiency. Automation, particularly in effect-based methods, has the potential to reduce manual intervention and make processes more sustainable.

Importantly, our work highlights the need to integrate green principles into analytical protocol development from the outset, addressing the current imbalance where environmental considerations are often secondary to analytical performance.

Do you anticipate similar results in using your technique to seek different chemicals and compounds?

Yes, I do think we get similar results when using these techniques to investigate methods for different chemicals and compounds. Many of the standards still in use today are quite dated, originating from a time when the primary focus was on achieving the highest analytical performance – which, of course, is still important. However, the growing emphasis on environmental sustainability has highlighted the need for modern approaches that balance precision with responsibility.

What difficulties did you encounter in your work, specifically sampling and analytical challenges?

In my prior practical work, I encountered several challenges already starting with the process of sampling and preparing water samples for endocrine disruptor analysis. A major difficulty arises from the growing need of monitoring anthropogenic pollutants in trace concentrations (pg/L) or as micropollutants (ng/L– μg/L). This necessitates an enrichment step prior to sample analysis. Solid-phase extraction (SPE) is commonly used to enrich water samples, but the complexity of environmental matrices and the low concentrations of target analytes require highly sensitive and selective analytical techniques. Even with state-of-the-art LC–MS/MS devices, it can be hard to achieve this in complex matrices. For example, if we consider the required environmental quality standard for 17alpha- ethinylestradiol (EE2) in surface water, which was on the watch list of the European Water Framework Directive, at a level of 0.035 ng/L. Achieving reliable quantification of such low concentrations presents a significant challenge, as it demands pushing detection limits even lower.

Despite advancements in sample preparation for bioassays, there are still challenges that need further optimization. Protocols for bioassays are still varying and do not address their specific requirements. One issue is that the actual toxicity of a sample can be altered during each step of the preparation process. For instance, during SPE protocols, which are mostly derived from chemical analysis, the chemical composition of the sample may change, with some active compounds being added or removed.

Lastly, regarding bioassays but also the identification of new compounds through non-target analysis, it is crucial not to exclude any compounds. However, SPE is not discrimination-free. What you extract is what you see and to capture a wide polarity spectrum with minimum discrimination is challenging to achieve. Nevertheless, as mentioned, without SPE, sample analysis would not be feasible either. Thus, we must continue to find a compromise.

How did you process the data to obtain the results you were looking for?

First, I familiarized myself with the metric tools to understand what they offer and how to use them. Some tools provide a dropdown menu with predefined answers, while others require you to enter specific numbers or assign scores based on your own judgment. According to the requirements, I made detailed lists of the analytical methods I wanted to evaluate, documenting the steps involved, solvent usage, consumables and reusable materials. I used established standards but also benefited from my own practical experience over the past few years working on endocrine disruptors in aquatic environments. For future scenarios we considered, based on published innovations in research and development, to apply these improvements to our initial list where possible. While these innovations are not yet incorporated into standards nor used in such combinations, we aimed to create potentially greener scenarios by integrating these advancements. Ensuring consistency by using the same two tools across all methods, allowed us to gain a clear comparison of EDC analysis and its potential for improvement.

Were there any factors that might affect the accuracy of your findings?

Even though the analysis has been conducted as objectively as possible, relying on facts and data, there remain always uncertainties that could affect the accuracy of research findings. In our study, these include variability in the data input for the assessment tools, as some values, such as energy consumption, were based on average estimates. In order to improve this in the future, we have established a system within the NACH-LABS project (Project 38337/01) based on smart home technologies, enabling detailed monitoring of energy consumption. This pilot setup will be expanded to include additional systems, further enhancing the data and helping identify potential savings in the context of greener analytics. Furthermore, the preset options in the tools did not always perfectly align with the specifics of the methods being considered, requiring some degree of approximation. The inherent limitations of the metric tools themselves and the complexity of the methods being assessed could also contribute to discrepancies in the results.

How do you imagine the results of your study can/will be applied?

I hope the results of my study motivate more innovations in (bio)analytical chemistry and their applied standards. The development of less hazardous chemicals and the advancement of technologies for monitoring contaminants are critical to a sustainable future. I also hope that this study serves as another example of how these tools can be applied and, ideally, will be used more and more frequently in research studies.

Long-term success requires fully integrating greener practices into a cohesive strategy that not only addresses current issues but also anticipates future risks. A paradigm shift is necessary – one that prioritizes safety and sustainability in the design and use of chemicals from the outset.

Are there any next steps in this research?

In our study, we found metric tools to be highly convenient and effective. We chose two tools that aligned best with our specific research objectives, but there are of course other options available. These tools are very versatile and I think can serve as valuable additions to many studies. They provide valuable insights, helping researchers gain a more comprehensive understanding of their approaches and their impacts. By providing specific scores, they also make results more comparable, which is an important step toward continuous improvement and refining methods for better efficiency and sustainability.

I certainly aim to continue using these tools in future projects to further enhance the awareness and integration of more environmentally sustainable analytical practices.

References

1. Klein, M.; Klassen, M. D.; Schmidt, T. C.; Tuerk, J. Integrating Green and White Analytical Principles for Endocrine Disruptor Analysis in Aqueous Matrices: From Routine Methods to Emerging Technologies. Green Analytical Chemistry 2025, 12, 100186. DOI: 10.1016/j.greeac.2024.100186

Since October 2020, Michelle Klein has been working as a Scientific Associate in the Department of Environmental Hygiene & Pharmaceuticals at the Institut für Umwelt & Energie, Technik & Analytik (IUTA) e. V., Duisburg, Germany. Since January 2023, she has also served as a Surrogate Quality Manager under ISO 17025.Michelle Klein is in the final stage of pursuing her doctoral degree, focusing on the ‘Analysis of Endocrine Disrupting Compounds and Their Effects in Aquatic Environments’ under the supervision of Prof. Dr. Torsten C. Schmidt at the Faculty of Chemistry, University of Duisburg-Essen. She holds a Master of Science in Environmental Toxicology from the University of Duisburg-Essen and a Bachelor of Science in Bioengineering (Rhein-Waal University of Applied Sciences, Kleve, Germany). Photo courtesy of Klein.