A review on environmental isotope analysis of aquatic micropollutants: Recent advances, pitfalls and perspectives
Introduction
In Europe, groundwater is the most important drinking water resource for many countries; e.g. up to 100% in Denmark or Austria, 80% in Switzerland and around 70% in France [1]. Although essential, pressure on water resources by human activity is increasing: organic micropollutants (OMPs) originating from agricultural, industrial or domestic use may reenter the water cycle (i) after inefficient wastewater treatment, (ii) from landfill leachates, or (iii) as residues from pesticides and nitroaromatic explosives. These OMPs can reach the different water bodies via surface runoff or by leaching and infiltration to groundwater. An unambiguous assignment of bio-/or chemical degradation pathways for these compounds is crucial to correctly assess the risk of soil and water contamination and to reliably estimate contaminant fluxes in the environment [2]. Furthermore, degradation metabolites can arise from multiple precursors, and a way to identify the origin of these compounds would be welcome. This is for example the case for the herbicides atrazine, cyanazine and simazine that can all give rise to deisopropylatrazine, a transformation product of concern in aquatic ecosystems. To the questions revolving around degradation behavior of OMPs and their environmental fate in wastewater and receiving water bodies, one can add a more forensic oriented view. For example, the environmental monitoring of explosive residues or illicit drug compounds and their precursors and degradation products in urban wastewater could provide useful information regarding clandestine activity in specific areas [3]. Isotope approaches can help providing answers to these questions, demonstrated by recent research on micropollutants and on legacy compounds at contaminated sites [[4], [5], [6]].
Characterizing the isotopic composition of organic pollutants in water involves three consecutive steps: 1. sampling and sample preparation, 2. the analytical process (measurement), and 3. data evaluation including referencing strategies (Fig. 1). Sampling and sample preparation is a crucial step to ensure accurate isotope ratio measurements, especially for the extraction of micropollutants from environmental samples [7,8]. Early isotope analysis of pesticides and other micropollutants involved labor-intensive offline compound extraction techniques, especially if the target compound was not present as pure substance. Isotope ratios were then measured with dual-inlet isotope ratio mass spectrometry (IRMS) or by using elemental analyzer-based techniques (EA-IRMS). These first studies aimed at establishing a multi-elemental isotopic database of agro-chemical formulations or in focused on source distinction [[9], [10], [11]]. From these early “bulk” stable isotope techniques (BSIA), focus switched relatively quickly to more convenient compound-specific isotope analysis (CSIA). CSIA has the advantage of enabling isotope ratio analyses of individual compounds within a mixture by online coupling of either gas or liquid chromatography with isotope ratio mass spectrometry (GC-IRMS and LC-IRMS). CSIA is nowadays not only one of the key techniques for identifying the origin of organic contaminants in the environment, but also for characterizing their transformation processes and degradation pathways. Degradation of pollutants involves biotic and/or abiotic transformations, bacterial enzymatic and/or (photo)chemical reactions, linked to measurable isotope fractionation effects. This isotope fractionation is a result of the slightly different reaction rates when breaking chemical bonds in molecules between atoms of only light isotopes of an element (lE) versus those involving a heavy isotope of an element (hE) in a reactive position. Such kinetic isotope effects hence serve as an indication for reactive processes, from which the pathway and extent of pollutant degradation can be derived [5,12]. Since these variations of isotope ratios are often small, they are reported in per mil (‰) using the δ-notation, where the isotopic composition of an element within a sample is expressed relative to an international isotope reference standard (Fig. 1). The Rayleigh equation (Fig. 1) links concentration changes, the fraction of the contaminant remaining after a time t, to changes of the stable isotope ratio of the degraded compound via an enrichment factor ε, determined in laboratory batch experiments. The slope (Λ) obtained by dual isotope plots (Fig. 1) allows for the identification of biodegradation pathways. For further information on the principles of isotope ratio analyses of organic compounds, and their application in environmental biogeochemistry, we refer to relevant CSIA-reviews that have been published over the last years [2,4,5,[13], [14], [15], [16], [17], [18]].
Some of these reviews focus on more microbiological viewpoints, the biochemical reactional processes and isotope fractionation patterns involved in degradation [2,5,13]. Others place special emphasis on the concept of 2D- or multi-element CSIA, where at least two elements are considered simultaneously as a better means to decipher the various, sometimes competing, biotic enzymatic and abiotic chemical degradation processes [[16], [17], [18], [19]]. Several reviews highlight the developments regarding the evaluation of in situ degradation of typical legacy pollutants in contaminated aquifers [15,16,18]; Nijenhuis et al. focus in addition on data interpretation of new isotope systematics and referencing strategies [17]. Before evolving in recent years into a routine approach for investigating bioremediation of common industrial legacy contaminants, CSIA had to already overcome some initial analytical pitfalls [20,21]. Although nowadays CSIA is a well-established analytical approach in environmental monitoring, its main application field is still rather limited to point-source pollution resulting from major industrial activities. Monitoring environmental degradation of OMPs and residues from non-point pollution sources is generally a difficult task due to diffuse inputs from human activity. In this context, some CSIA-reviews focused on OMPs such as agrochemicals, pharmaceuticals, and nitroaromatic compounds [4,14,17,22]. These reviews shed some light on the challenging analytical aspects associated with CSIA applications of micropollutants such as pesticides and highlighted the need of future research towards advanced integrative environmental studies, especially on the catchment scale. Indeed, in the last years a lot has been done to improve performance of routine CSIA for these molecules. Despite important technological progress, analytical uncertainties related to low concentrations, typical for non-point pollution sources, combined with only subtle isotopic changes, often observed during degradation of environmental micropollutants such as pesticides, call for a critical assessment of these developments. In the present review, we provide a comprehensive overview on the latest technical developments concerning OMPs that can be encountered in a context of diffuse pollution in wastewater, surface waters or groundwater: pesticides, industrial compounds, pharmaceuticals and personal care products (PPCPs), and nitroaromatic compounds including organic explosives (NACs/OEs). Using a reference database of nearly 200 CSIA-studies of OMPs (Appendix A in Supplementary Material), we describe the analytic challenges and address common pitfalls, often overlooked in previous review works. Our main focus is to provide an up-to-date compilation of the most recent technical developments in this field.
The first CSIA-studies on OMPs using GC-IRMS include work on nitroaromatic compounds such as the explosive trinitrotoluene or substituted nitrobenzenes [7,23,24]. Concerning pesticides, first GC-IRMS methods were developed for the herbicide compounds isoproturon and atrazine [[25], [26], [27]]. Early CSIA-studies on industrial micropollutants and PPCPs dealt with chlorinated benzenes [28,29], dioxins [30], phthalates and synthetic musk compounds [31]. OMPs in fresh water environments can have a range of different physico-chemical properties; the analytical methods that have to be used depend on their degree of volatility and polarity [32]. Emerging contaminants are often too involatile or too polar for GC analysis and cannot be purified in sufficient quantities for EA-IRMS characterization. Although the first micropollutant isotope studies using LC-IRMS were published a decade ago [33,34], it has not become a routinely applied technique yet. Our survey over the last 20 years of the published literature on isotopic analyses of the targeted OMP classes (pesticides, industrials, PPCPs and NACs/OEs, Fig. 2) shows a predominant focus on volatile and semivolatile nonpolar contaminants where GC is the analytical separation technique of choice (>165 out of nearly 200 CSIA applications, <15 using EA- or LC-IRMS; cf. Fig. 3, Appendix A, fig. A.1). Non-volatile compounds or high polarity contaminants require in general LC-IRMS or can only be studied using GC-IRMS after derivatization: transformation of the analytes to more volatile species. Contrary to “classic” organic pollutants which consist mostly of carbon and hydrogen, OMPs can contain a significant amount of nitrogen, chlorine, phosphorus, bromine or sulfur within their structure and incomplete combustion may cause method-induced isotope fractionation [35,36]. Furthermore, early examples as well as very recent CSIA-studies using GC-IRMS on the catchment scale revealed insufficient chromatographical resolution and low environmental concentrations as one of the main restrictions of CSIA-analyses in natural surface waters [31,37]. Besides the need for highly efficient sample preconcentration and optimal chromatographic resolution of the molecules of interest, their conversion into analyzable gases under continuous flow conditions, i.e. online combustion or high-temperature reduction for IRMS instruments, is critical for precise and accurate isotope analyses [14,20,21]. The most work on micropollutant CSIA has been done for the carbon isotope systematic (Fig. 3), followed by nitrogen, hydrogen, and chlorine. Especially the analysis of the latter two elements require innovative and advanced isotope techniques. Methods exist for isotopic ratio measurements of other elements, recently including bromine or sulfur, and first Br-/S-CSIA studies have become available [12,17,[38], [39], [40]]. So far, Br- and S-CSIA have not been widely applied and are mainly performed on GC-MC-ICPMS instruments and, hence, are not included in the present review. The following sections will present a critical review of the aforementioned technical problems for compound-specific isotope analysis (CSIA) of OMPs, common pitfalls in instrumental approaches and ways to avoid them. Our review especially focusses on possible implications of the analytical process on instrumental accuracy and precision (section 2). Section 3 sheds light on how to improve method detection limits for CSIA by sample preparation techniques for water samples to achieve the required concentration of OMPs.
Section snippets
GC-interface designs for C- and N-isotope analysis
Since the introduction of commercial GC-IRMS interfaces, the components in the combustion interface for C- and N-isotopes were optimized to minimize peak broadening: (i) reducing GC effluent pathway dimensions and (ii) dead volumes, and (iii) providing leak-free connections. Interferences due to combustion byproducts (NOx, SOx, H2O) can impair accuracy and precision. H2O is removed by an on-line system using a water permeable polymeric capillary (Nafion™ membrane). Any NOx produced in the
Challenges in sample preparation and perspectives proposed by recent developments
IRMS instruments are non-scanning magnetic sector mass spectrometers where the ions are focused on dedicated, mass-specific Faraday cup collectors. IRMS detectors are designed to measure variations at natural isotopic abundance levels with a very high precision and accuracy of 0.0001 atom%, which translates in a difference of 0.1‰ in the δ isotope ratio notation. However, the measurement of these subtle differences in organic compounds comes at a cost: IRMS detectors are, compared to molecular
Summary and future research needs
The main advantage of CSIA is that it delivers degradation-related information based on isotopic fractionation of the parent compound. Thus, it provides direct evidence of degradation even in the absence of detection or analysis of known metabolites, or if the metabolites are not known. A big drawback in the context of diffuse inputs is that multiple water flow and solute transport pathways lead to mixed isotopic signatures. To date, most of the studies deal either with development of new
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors are grateful to G. Grimm for language and style editing of the manuscript and for his great help with the illustrations. The project received financial support from the Research Program “GESTEAU” of BRGM, the French Geological Survey.
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