Analytical approaches to uncover the hidden identity of non-extractable organic soil contaminants - a synopsis

Environmental fate assessment of organic contaminants in soil is essential for chemical risk assessment within the regulatory frameworks. While conventional biodegradability testing allows quantification of extractable residues and mineralization, the analysis of non-extractable residues (NER) remains a significant analytical challenge. This review synthesizes current analytical strategies and workflows for the characterization and differentiation of NER in soil originating from organic contaminants. Therefore, we describe current analytical strategies and workflows that enable robust NER characterization. The analytical framework distinguishes three NER types based on formation mechanisms and environmental relevance: Type 1 (physically entrapped residues sequestered within soil aggregates with remobilization risk), Type 2 (covalently bound residues with strong chemical bonds to a solid matrix and minimal remobilization potential), and Type 3 (microbial biomass residues integrated into natural organic matter with no environmental concern). Sequential extraction procedures using aqueous and organic solvents, combined with targeted chemical degradation methods and derivatisation like silylation, provide systematic approaches to distinguish NER types. Silylation enables the release and quantification of physically entrapped (Type 1) NER, while remaining covalently bound (Type 2) NER require chemo- or thermochemolytic cleavage for identification. Biogenic NER (Type 3) are quantified via acidic hydrolysis and analysis of the released amino acids as microbial biomass proxies carrying the label from the degraded contaminant. Isotopic labelling utilizing both radioactive ( 14 C, 3 H) and stable ( 13 C, 15 N, 2 H) isotopes has greatly advanced mass balance tracking and molecular identification capabilities. While radioactive labelling provides unparalleled sensitivity for quantification, stable isotope methods coupled with GC-IRMS, LC-MS/MS, or NMR offer mechanistic and structural information. So far, NER research using isotope labelling was mainly based on 14 C-, 13 C- and 15 N, but recently the suitability of 2 H-labels for this purpose has been described. This innovative approach, which can ease distinction between the formations of xenobiotic NER (type 1 and 2) and biogenic NER (type 3), is presented here. By integrating state-of-the-art extraction, degradation, and isotope-tracing techniques, researchers can now achieve robust discrimination of NER types. These analytical advancements are crucial for regulatory bodies to assess chemical safety, support persistence classification, and predict long-term risks associated with organic pollutants in soils. Suggestions for further research on the use of Deuterium and Tritium as isotope labels and the investigation of mass-dependent isotope effects on the degradation of the labelled test substance are also given. • Reviews state-of-the-art analytical methods for characterizing non-extractable residues (NER) in soils. • Distinguishes three NER types based on environmental relevance: Type 1 (physically entrapped), Type 2 (covalently bound), and Type 3 (biogenic). • Summarizes extraction, derivatization, and chemical degradation techniques for NER differentiation. • Compares radioactive and stable isotope labeling approaches for mass balance tracking and structural identification. • Emphasizes the importance of NER characterization for chemical persistence (P) assessment under regulatory frameworks. • Provides guidance on integrating isotope labeling with advanced analytical techniques (e.g., GC-IRMS, LC-MS/MS, NMR). • Suggests future research directions, including the application of Deuterium and Tritium and isotope effect studies.