AOX Rapid Assessment

Rapid Assessment of Adsorbable Organically Bound Halogens (AOX) in Waters Determination of AOCl, AOBr, AOI and AOF by Combustion Ion Chromatography (CIC) according to DIN 38409-59

Introduction

Adsorbable organically bound halogens (AOX) represent a complex index encompassing the sum of numerous halogenated organic compounds that can be adsorbed on activated carbon. Historically, this total AOX parameter has been determined via microcalorimetric titration according to DIN EN ISO 9562 or EPA 1650. By definition, AOX comprises adsorbable organically bound chlorine (AOCl), bromine (AOBr) and iodine (AOI) that cannot be individually determined, but does not include fluorine (AOF). Many of these organic halogen compounds and their degradation products pose serious risks to human health and the environment due to their toxic, mutagenic and carcinogenic properties, their ubiquitous distribution, their persistence and their bioaccumulation in the food chain.

Origins and Sources

The concept of AOX as a total parameter was introduced in 1976 as a means of rapidly measuring organohalogenated compounds in wastewater, drinking water and natural water sources. The concept was subsequently expanded, termed "AOX-S18", to include certain insoluble compounds in wastewater treatment sludge and precipitates. Naturally derived halogenated organic compounds were first isolated in 1896, with their numbers increasing following the development of suitable separation and identification techniques. To date, over 5,000 naturally derived organohalogens have been discovered, predominantly organochlorine (50.6%), organobromine (44.8%) and in smaller proportions organoidine and organofluorine (4.6%). While some organohalogens are naturally produced by living organisms with relatively low adverse effects, those produced through industrial and other anthropogenic pathways are widespread and persistent, and thus capable of causing much greater negative impacts. From the second half of the 18th century onwards, the Industrial Revolution gained momentum, and the widespread production of halogenated organics expanded, encompassing not only chlorinated hydrocarbons used for agricultural, industrial and domestic purposes, but also other organic compounds such as PCBs (polychlorinated biphenyls), DDT (dichlorodiphenyltrichloroethane) and atrazine. During the 1980s, public awareness of the negative environmental impacts of AOX began to rise steadily. The primary driver was the discovery of chlorinated organics and dioxins in pulp and paper mill waste, though the tragic consequences of the Seveso disaster and the long-term effects of "Agent Orange" used in the Vietnam War also played important roles. The primary anthropogenic sources of AOX are industrial emissions such as wastewaters from pulp and paper producers, the textile dyeing and finishing industry, and production facilities manufacturing synthetic organohalogens and their end products (e.g., flame retardants or PCBs). Drinking water disinfection processes, wastewater treatment plants and municipal sewage networks are also significant sources of AOX. Agricultural runoff represents a widespread and difficult-to-treat AOX source, containing a range of herbicides, insecticides and pesticides (e.g., atrazine and DDT [Stockholm Convention, Germany's national DDT legislation]) many of which are banned in different countries. In hospital wastewater, organically bound iodine, particularly iodinated X-ray contrast agents, pharmaceuticals and disinfectants such as triclosan also contribute to AOX levels. In recent years, particular attention has been paid to organofluorine compounds known as "forever chemicals", such as PFAS (per- and polyfluorinated alkyl substances). This class, comprising over 10,000 chemicals, is persistent and bioaccumulates in the environment as much as in the human body. Many PFAS pose broad risks to both the environment and human health, and unfortunately, targeted analysis of certain compounds in this class is time-consuming, complex and expensive. Conversely, the determination of adsorbable organically bound fluorine (AOF) in water samples and extractable organically bound fluorine (EOF) in solids and biological matrices as total parameters has been widely adopted by the scientific community for investigating and monitoring organofluorines. The general formation and degradation cycle of AOX is complex. Figure 1 depicts the widespread distribution and diverse transport of some organic halogens.

Regulations

As a total parameter, AOX provides a valuable solution for laboratories in assessing, identifying, monitoring and controlling sources and sink areas, as well as discovering the efficiency of removal technologies for organohalogens. For example, organohalogens are listed as potential pollutants in the German Water Management Act, based on the EU Water Framework Directive (2000/60/EC). AOX is directly regulated in the German Wastewater Ordinance (AbwV) and the German Wastewater Tax Act (AbwAG) for wastewaters, and in the German Sewage Sludge Ordinance (AbfKlärV) for solid wastes. AOX discharge thresholds have been set at 100 μg/L or 10 kg/year (AbwAG), while the treatment sludge limit is fixed at 400 mg/kg dry weight (DIN 38414-18, DIN 16166). Wastewater treatment requirements vary for specific industries and are regulated within the AbwV framework by maximum concentration values as well as annual load values.

Development of Analytical Methodology

To date, all requirements used for AOX measurement in water and wastewater samples have been based on the analysis method containing AOCl, AOBr and AOI presented by Kühn in 1974. In this method, organically bound halogens in acidified samples are concentrated on activated carbon, while inorganic halogens are rinsed with an acidified nitrate solution, and finally, following a mineralization step (combustion), adsorbed organically bound halogens are detected by microcalorimetric method. The high affinity of chloride, bromide and iodide for silver cations produced during titration with silver nitrate causes the formation of precipitates and permits determination of the total AOX expressed in terms of chloride. However, silver fluoride complexes are highly soluble and therefore this value is not accounted for in any AOX parameter determination according to Kühn or in the standardized procedure for water samples specified in DIN EN ISO 9562. A similar approach is found in EPA 1650. Titration of mineralized organohalogens adsorbed on activated carbon allows determination of AOX only in terms of chloride, while analysis by IC following pyrohydrolytic combustion facilitates separate determination of chloride, bromide and iodide as well as fluoride. This new method, a promising approach developed for the determination of organically bound fluorine, chlorine, bromine and iodine (AOF, AOCl, AOBr and AOI) parameters, involves mineralization of samples via automated combustion followed by analysis with Ion Chromatography (CIC). This method has been standardized in the recently published DIN 38409-59. In this standard, individual AOX types can be resolved (e.g., to investigate AOX sources and control specific treatment processes), while the AOF total parameter can also be used to monitor PFAS. The CIC-AOX_(Cl) total parameter (expressed in terms of chloride) can be evaluated by summing the values of AOCl, AOBr and AOI (with appropriate mass correction). Due to the novelty of this approach, CIC-AOX_(Cl) is not yet equivalent to AOX as defined in past regulations.

Experimental Approach

Metrohm, together with Analytik Jena's automated sample preparation unit (APU sim), offers a fully automated analytical solution that can meet CIC-AOX (Cl) specifications via AOCl, AOBr and AOI according to the new DIN 38409-59 standard and also enables AOF determinations. The general working principle of the method (Figure 2) involves concentration of AOX on activated carbon, followed by automated combustion and separate detection of halogens by IC. Without any additional effort, the total parameters of AOX and AOF can be determined from the measured halogen concentrations. On the other hand, there are minor differences in sample preparation procedures for AOX (AOCl, AOBr and AOI) and AOF determinations (according to DIN 38409-59). The advantage of the new DIN standard is that it permits determination of not only AOF but also individual total parameters of adsorbable organically bound halogens. Sample Preparation: Adsorption of Organohalogens The general sample preparation procedure (pre-concentration and adsorption of organohalogens) is similar to DIN EN ISO 9562, and adsorption on activated carbon is the key point in both methods. In AOF determination, samples having a neutral pH is of great importance to prevent inorganic fluorine adsorption onto activated carbon, while for measurement of other organically bound halogens, acidification of samples (similar to DIN EN ISO 9562) is mandatory. For AOX (e.g., AOCl, AOBr and AOI) determination, samples must be acidified to pH<2 with nitric acid prior to the pre-concentration step (Table 1). On the other hand, since AOF determination is also covered within DIN 38409-59, these samples must be subjected to a neutralization process. This requirement is met with the addition of sodium nitrate. Adsorption of organically bound halogens is performed as an automated sample preparation step within Analytik Jena's APU sim system (Figure 2). Despite the general procedure containing several steps, automation of up to six samples and parallel processing makes this stage a standardized preparation method with perfect repeatability and high sample throughput of approximately 60 samples per working day (less than 45 minutes per batch of 6 samples). In summary, two columns filled with activated carbon connected in series (at least 50 mg per column) are rinsed with 100 mL sample at a flow rate of 3 mL/min. While organically bound halogens are adsorbed onto activated carbon (disposable columns specifically used for AOF and AOX determination, see Table 1), inorganic halogens are rinsed with an additional 25 mL wash solution. Following the automated sample preparation process, the entire contents of both columns are transferred to two separate ceramic crucibles for CIC analysis (not mandatory; a single crucible can also be used for combustion).

Combustion IC Analysis

Following transfer of activated carbon to ceramic crucibles, each crucible is combusted at 1050°C in the presence of argon and oxygen gas flow using a combustion module (Figure 2). Combustion is performed pyrohydrolytically, and the use of a water stream to convert halogens to their hydrogenated forms is important during this process. For this purpose, addition of ultra-pure water at a rate of 0.2 mL/min is performed using Metrohm's automatic and precise liquid handling tool, Dosino, during the combustion step. The automated sample handling features provided by the system minimize manual laboratory work for users, allowing analysis of up to 35 samples in succession. During the combustion step, organohalogens contained in the 100 mL sample adsorbed on activated carbon are vaporized and then transferred to the absorption solution (ultra-pure water) by an Ar/O2 gas stream, being transferred to the liquid phase. With the aid of additional Dosino units, the sample that has entered the aqueous phase is transferred to the IC instrument for analysis. In addition to fully automated combustion, sample absorption and liquid handling steps, further automation features can increase the efficiency of Metrohm CIC for AOX analysis: • 941 Eluent Preparation Module, enabling automatic eluent generation and near-unattended operation of the IC system. • Metrohm Intelligent Partial Loop Injection Technique (MiPT), allowing both automatic calibration and variable volume (4-200 μL) sample injection adjustable based on the halogen content of the sample. • Metrohm Intelligent MagIC Net software, in addition to the usual device control and data processing features of chromatographic data software programs, enables logical and nested operation. With high-low calibration applied, results are automatically assigned to the appropriate calibration range and correct data calculation is guaranteed, eliminating the need for further sample dilution work. In the ion chromatography system, separation of halogens is performed using a Metrosep A Supp 5 - 250/4.0 column and A Supp 5 Guard/4.0 guard column. AOF (F-) is eluted from the column in less than seven minutes, while analyte peaks for AOX (Cl-, Br- and I-) are completely obtained in less than 25 minutes (Figure 3B). Using the MiPT technique, automatic system calibration is performed using inorganic anion standards for fluoride, chloride, bromide and iodide (1 g/L standard solutions, Sigma-Aldrich TraceCert®, Table 2). To control the overall performance of AOF and AOX determination, organic reference standards with different concentrations are used (4-fluorobenzoic acid: 3-19 μg/L AOF, 4-chlorobenzoic acid: 20-90 μg/L AOCl, 4-bromobenzoic acid: 2-9 μg/L AOBr and 4-iodobenzoic acid: 2-9 μg/L AOI). To ensure validation of the general procedure is checked, these standards are prepared in the same manner as samples: pre-concentration on activated carbon is provided using APU sim and subsequently automatically analyzed with the CIC system. Unlike halogen and sulfur analysis in aqueous samples, the procedure for AOX and AOF is somewhat more complex. Special sample vessels and special activated carbon (e.g., fluorine-free materials for AOF determination, Table 2) are required to ensure low background and prevent cross-contamination. However, blank (blank) measurement readings (Equation 1) are required for samples subjected to pre-concentration of ultrapure water to provide correct and reliable results for CIC-AOX(Cl) and AOF for proper and reliable results.

Analysis Results

Individual concentrations for AOCl, AOBr and AOI as well as AOF in neutralized samples are calculated according to Equation 1. Results for each of the two adsorption columns are summed separately and corrected for column blank values and relevant volumes. Despite the need for special materials, sensitive analysis of halogens with suppressed conductivity detection and complex sample preparation procedures, blank values are quite low and were only measurable for fluoride and chloride (Table 3). Overall detection limits (LOD values based on calculations in DIN 32645) are below 0.5 μg/L for AOBr, AOI and AOF and approximately 1.4 μg/L for AOCl. LOD values are calculated using the calibration curve for AOBr and AOI as no blank value was detected, while for AOCl and AOF they are calculated according to the blank method (Table 3). The described approach is seen to conform to and even exceed the sensitivity of the DIN 38409-59 standard. During DIN validation, a series of water samples were analyzed by different laboratories using a system configuration similar to that described in this technical article. The full data set, published on the Water Chemistry Society website (wasserchemische-gesellschaft.de) after publication of the DIN standard, demonstrates the validity and robustness of the general procedure as a routine method. Results for three selected samples and a control standard are shown in Figure 3A. In each case shown in Table 3, four witness samples were prepared and analyzed. The CIC-AOX(Cl) content (as the mass-corrected sum of AOCl, AOBr and AOI) spans the range of 46-120 μg/L. Higher values correspond to Wastewater #2 and surface water sample and demonstrate the diversity of pollution sources (Figure 3A). By using the IC system as an analytical technique, it is now possible to measure not only the CIC-AOX(Cl) total parameter but also the individual fractions that contribute to the AOX content. In all water types, AOCl constitutes the largest portion of the AOX fractions (85-90%), while contributions of AOI (21-35%) and AOBr (4-10%) are less dominant. AOF contents vary between 7-9 μg/L (Figure 3A). RSD values for replicate analyses are obtained below 5%, demonstrating the excellence of method performance. This simple, fast and reliable procedure permits monitoring of PFAS (as AOF) as a first step before employing more complex analytical techniques (e.g., LC-MS/MS or GC-MS) for a more detailed view of individual PFAS.

Conclusion

The world of organohalogens is so diverse that measurement of total parameters provides a simple means of gaining much more information about the sources, transport pathways and particularly about sensitive areas of these compounds. Complex targeted analytical methods (e.g., using LC-MS/MS or GC-MS) can resolve certain individual halogenated organic compounds in a secondary determination stage for deeper investigations. With the new DIN 38409-59 standard, a reliable and fast method is provided for correctly determining the CIC-AOX_(Cl) total parameter and AOCl, AOI and AOBr fractions as well as AOF. Standardization of the adsorption procedure with the APU sim unit with high sample throughput (six samples in parallel) and fully automated CIC analysis (including automatic calibration with MiPT and automatic data calculation) makes this technique an ideal solution for routine laboratory analyses. The high-sensitivity detection of separate AOX fractions and AOF extends the standardized procedure without any additional work to monitor AOF and investigate separate sources of AOX. Overall, the entire validated procedure embodies key advantages such as simple, easy and standardized operation, precise determination of analytes, automatic calculation of results, and a complete low-maintenance system available from a single supplier. The fully automated nature of the method increases the repeatability, accuracy and reliability of results. This also provides valuable savings in laboratory time related to liquid handling, standard and eluent preparation processes, while enabling 24/7 analyses from which any laboratory—whether research, routine or government—can benefit. References 1. Metrohm White Paper: WP-081EN-2022-10 "Fast assessment of adsorbable organically bound halogens (AOX) in waters" 2. Metrohm White Paper: WP-078EN – 2021-12 "Adsorbable organic fluorine (AOF) - a sum parameter for non-targeted screening of per and polyfluorinated alkyl substances (PFASs) in waters" 3. Metrohm Application Note: AN-CIC-033 "Monitoring PFASs in water sources – Non targeted adsorbable organically bound fluorine(AOF) analysis by CIC" 4. Metrohm Application Note AN-CIC-034 "Fast analysis of AOX in waters by CIC Measurement of AOCl, AOBr, AOI, and AOF according to DIN 38409-59" 5. Metrohm Blog Article "CIC analysis of halogenated organic compounds according to DIN 38409-59" https://www.metrohm.com/tr_tr/discover/blog/20-21/analyzing-halogenated-organiccompounds-with-cic-according-to-di.html     Elif Metin Kulaksız Ion Chromatography Product Group Manager Metrohm Turkey