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Carbon, Hydrogen and Chlorine Stable Isotope Fingerprinting for Forensic Investigations of Hexachlorocyclohexanes Natalija Ivdra,†,‡ Anko Fischer,‡ Sara Herrero-Martin,†,§ Thomas Giunta,∥ Magali Bonifacie,∥ and Hans-Hermann Richnow*,† †

Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research - UFZ, D-04318 Leipzig-Halle, Germany Isodetect GmbH, D-04103 Leipzig, Germany § Institute of Landscape Biogeochemistry, Leibniz-Centre for Agricultural Landscape Research (ZALF) e.V., D-15374 Müncheberg, Germany ∥ Equipe Géochimie des Isotopes Stables, Institut de Physique du Globe de Paris, Sorbonne Paris Cité, Université Paris Diderot, UMR 7154 CNRS, 75005 Paris, France ‡

S Supporting Information *

ABSTRACT: Multielemental stable isotope analysis of persistent organic pollutants (POPs) has the potential to characterize sources, sinks, and degradation processes in the environment. To verify the applicability of this approach for source identification of hexachlorocyclohexane (HCHs), we provide a data set of carbon, hydrogen, and chlorine stable isotope ratios (δ13C, δ2H, δ37Cl) of its main stereoisomers (α-, β-, δ- and γ-HCHs) from a sample collection based on worldwide manufacturing. This sample collection comprises production stocks, agricultural and pharmaceutical products, chemical waste dumps, and analyticalgrade material, covering the production time period from the late 1960s until now. Stable isotope ratios of HCHs cover the ranges from −233‰ to +1‰, from −35.9‰ to −22.7‰, and from −6.69‰ to +0.54‰ for δ2H, δ13C, and δ37Cl values, respectively. Four groups of samples with distinct multielemental stable isotope fingerprints were differentiated, most probably as a result of purification and isolation processes. No clear temporal trend in the isotope compositions of HCHs was found at the global scale. The multielemental stable isotope fingerprints facilitate the source identification of HCHs at the regional scale and can be used to assess transformation processes. The data set and methodology reported herein provide basic information for the assessment of environmental field sites contaminated with HCHs.

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INTRODUCTION Hexachlorocyclohexanes (HCHs) are among the most relevant persistent organic pollutants (POPs).1 These compounds are of high environmental concern because of their global distribution, bioaccumulation, and persistence, as well as their toxicity for environmental and human health.2−5 HCHs, mainly γHCH, have been broadly used worldwide since 1942 as agricultural insecticides leading to the widespread contamination of soils,6 as well as aquifers and the atmosphere as a result of dissolution and volatilization.7,8 γ-HCH is synthesized by the chlorination of benzene with molecular chlorine under free-radical conditions.9 In this reaction, a mixture of stereoisomers (technical HCH, t-HCH) is produced, containing mainly five different HCH stereoisomers with 60−70% α-HCH, 10−15% γ-HCH, 5−12% βHCH, 6−10% δ-HCH, 1−2% ε-HCH, and about 1−2% other isomers and impurities.9,10 Although γ-HCH is the only isomer showing strong insecticidal properties, t-HCH was applied for crop protection for many years. To avoid the adverse © XXXX American Chemical Society

environmental and human health effects and the unpleasant smell of the stereoisomers without insecticidal properties,6 tHCH was replaced in 1970 in Western Europe and the United States by enriched γ-HCH (typically 40% in the isomeric mixture)9 and later, starting from 1979 by purified γ-HCH, also called lindane when the purity is ≥99%. The application of lindane made necessary the development of advanced isolation and purification procedures for the production of γ-HCH with a high purity at lower costs. The isolation and purification strategy for the production of lindane is highly inefficient because of the generation of large amounts of chemical waste [Supporting Information (SI) section 1]. For each ton of lindane, 8−12 tons of other HCH isomers are produced generally, which have been dumped Received: Revised: Accepted: Published: A

June 17, 2016 November 7, 2016 November 22, 2016 November 22, 2016 DOI: 10.1021/acs.est.6b03039 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

conversion reactor for accurate continuous-flow hydrogen isotope analyses of chlorinated compounds by elemental analysis− (EA−) and gas chromatography−isotope ratio mass spectrometry (GC−IRMS), respectively. Additionally, a recent study by Gilevska et al.23 presents the optimization of an offline conversion method for chlorine isotope analysis by dual-inlet(DI-) IRMS, providing accurate and reproducible chlorine isotope ratios for HCHs. Application of these latest developments allowed us to overcome earlier analytical limitations for multielemental isotope analysis of chlorinated organic compounds. In this study, we aimed to evaluate the use of multielemental stable isotope analysis as a tool for assessing the sources, sinks, and degradation processes of HCHs in the environment. For this purpose, we have optimized state-of-the-art methods for hydrogen and chlorine stable isotope analysis. As a result, we provide the largest data set of carbon, hydrogen, and chlorine stable isotope ratios of HCH isomers for characterizing sources to date. The sample collection comprises commercially available analytical standards, chemical stocks of former producers, samples from chemical waste dumps (not affected by degradation processes), and agricultural and pharmaceutical formulations. The chemicals were produced in at least 15 different countries worldwide within the production period from the 1960s until now. This study provides valuable information for the interpretation of isotope fingerprints (13C/12C, 2H/1H, and 37Cl/35Cl) of HCHs with respect to production time, origin, and manufacturing process. Therefore, it can be used to investigate the sources, transport, degradation processes, and sinks of these toxic and persistent contaminants, whose global distribution and final fate still remain an issue.

worldwide, often in an uncontrolled and thoughtless manner, posing long-term environmental risks. The production and agricultural use of γ-HCH were banned in the European Union in 2000 and in the majority of other countries in 2009 through the ratification of the Stockholm Convention on POPs. Limited use of lindane for agricultural, medicinal, and/or veterinary purposes is still allowed in some countries, including the United States, Canada, Mexico, India, and New Zealand.11 To our knowledge, at this time, large-scale production is continuing officially only in India. Because of the environmental concerns related to HCHs, there is a high need for investigating their sources, potential sinks, and transformation processes. Variability in the stable isotope ratios of organic compounds, found both in originally manufactured chemicals and in environmental samples from contaminated sites, can be explained by isotope fractionation occurring during the synthesis, purification, handling, storage, application, and eventual degradation of these substances in the environment. Thus, stable isotope analysis is an efficient tool for tracking sources and sinks of pollutants in the environment, as well as assessing their transformation processes.12−14 Benzene, used as a starting material for HCH production (see SI section 1), is generally obtained from fossil sources with carbon isotope ratios in the range between −33‰ and −19‰13 and hydrogen isotope ratios between −97‰ and −28‰.15,16 Therefore, the relatively broad ranges of C and H signatures of the raw material and the analytical uncertainties in the available analytical methods (less than or equal to ±0.5‰ for carbon17 and less than or equal to ±5‰ for hydrogen18) open the possibility of distinguishing between different producers on the basis of the sources of raw material. Cl2 used for the production of HCH is generally produced by the oxidation of Cl− during electrolysis of NaCl by the mercury-, diaphragm-, or membrane-cell method from concentrated seawater chlorides (brines) or dissolved rock salt19 with chlorine isotope ratios within the narrow range between −0.5‰ and 0.0‰, equal or close to the standard mean ocean chloride (SMOC) reference value.20−22 Possible variations in chlorine isotope ratios in HCHs, which can be detected by available methods (with analytical uncertainty of less than ±0.1‰, 1σ),20,23 can arise from chlorine isotope fractionation during different technological processes of Cl2 gas production, as well as during the HCH synthesis and purification processes. These isotope effects can be especially pronounced for redox reactions, leading to high enrichment in 37 Cl of the most oxidized species.24 Therefore, the chlorine isotope ratio might serve as an additional descriptor for source investigations. To date, most of the studies aiming to track sources and fates of HCHs in the environment have focused on carbon isotope analysis.25,26 In several studies, carbon isotope ratios from different sources were reported.26−30 However, studies reporting hydrogen31 and chlorine23,29 isotope compositions of HCHs are much less frequent (Tables SI-3.1 and SI-3.2, SI). This gap is due to the analytical limitations on continuous-flow stable isotope analyses of hydrogen in chlorinated compounds and of chlorine, because of the formation of HCl, which is corrosive for the detector.32 The limited amount of isotope data for HCHs from different sources hampers the evaluation of the applicability of multielemental stable isotope analyses for the source identification and fate prediction of HCHs. New analytical methods were recently developed by Gehre et al.18 and Renpenning et al.,33 introducing a new Cr-based

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EXPERIMENTAL SECTION Solvents and Chemicals. Solvents including dichloromethane (DCM, ≥99.8%, Rostisolv), n-pentane (Pent, HPLCgrade, Rostisolv), and acetone (≥99.9%, UV/IR-grade, Rostisolv) were obtained from Carl Roth (Karlsruhe, Germany). The chemicals α-HCH (99.8%), β-HCH (98.2%, Fluka, Pestanal), δ-HCH (99.5%, Fluka, Pestanal), and γ-HCH (99.1%, Supelco, Pestanal) for the quantification of HCH isomers in noncommercial samples were purchased from Sigma-Aldrich Corporation (St. Louis, MO). Collection of Samples. Commercial samples of pure HCH isomers (Table SI-1.1) were obtained from the following companies: Aladdin Reagents Co., Ltd. (producer/supplier, Shanghai, China), Greyhound Chromatography and Allied Chemicals (supplier, Birkenhead, U.K.), Chem Service Inc. (supplier, West Chester, PA), Dr. Ehrenstorfer GmbH (producer/supplier, Augsburg, Germany), Sigma-Aldrich Corporation [producer/supplier, St. Louis, MO], with the brands Riedel-de-Haën (production site in Seelze, Germany), Fluka (production site in Buchs, Switzerland), Supelco (production site in Bellefonte, PA), Sigma-Aldrich India (production site in Bangalore, India), Himedia Laboratories Pvt. Ltd. (Mumbai, India). Lindane-containing pharmaceutical products (Table SI2.1), namely, Benhex cream (PSM Healthcare, Auckland, New Zealand) and PMS lindane shampoo (Pharmascience, Montreal, Quebec, Canada), were purchased through online pharmacies. Delitex shampoo (Delicia, Delitzsch, Germany) was obtained through direct contacts with the former producer. Noncommercial samples of HCH isomers and mixtures from B

DOI: 10.1021/acs.est.6b03039 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Hydrogen GC−Cr/HTC−IRMS Analyses for HCH-Containing Mixtures (Purity < 95%). The chromium-based continuous-flow conversion method was also employed for compoundspecific hydrogen isotope analyses using the recently developed method33 (GC−Cr/HTC−IRMS, SI section 8). In addition, we optimized the injection and flow conditions (injector’s temperature 300 °C, flow 1 mL/min) to achieve higher amplitudes and better peak shapes and also determined the precision and linearity ranges for individual HCH isomers. The results were calibrated against the δ2H values measured by EA− Cr/HTC−IRMS using three hexadecane standards and five laboratory HCH standards (SI section 8). Chlorine Isotope Analyses. Chlorine Offline DI-IRMS Analyses for Selected Pure Samples of HCH (Purity ≥ 95%). For chlorine isotope analyses, selected pure HCHs were first converted to methyl chloride (MeCl) by an offline sample preparation procedure20 (SI section 9) that we recently optimized and validated for HCHs.23 Then, δ37Cl values were measured by dual-inlet-isotope ratio mass spectrometry (DIIRMS). The overall precision of the analytical method applied in this study was ±0.1‰ (1σ, calculated by combining the reproducibilities of routine DI-IRMS analyses20,36,37 and the sample preparation method23). The accuracy of the method used in this study was confirmed by analyses of two subsamples of γ-HCH standard (labeled as P3, Tables SI-1.1 and SI-1.2) with a previously reported δ37Cl value (−0.18‰ ± 0.03‰, n = 3).23 Among others, samples with minimum and maximum δ13C and δ2H values were selected for the chlorine isotope analyses to represent the maximum possible range of carbon and hydrogen ratios in the third dimension. Graphical Software. The principal component analysis (PCA) function of SigmaPlot (version 13.0; Systat Software, Inc., San Jose, CA) was used to group samples with significantly different isotope compositions.

chemical production stocks, agricultural products, and chemical waste samples were obtained from various sources worldwide through personal contacts and then classified either as pure isomers (Table SI-1.1) or as mixtures (Table SI-2.1) according to purity analyses. Sample Preparation and Purity Analyses. Samples of commercially available analytical standards of pure HCH isomers were of analytical grade (purity ≥ 98%). Crystalline noncommercial pure-phase materials, highly concentrated chemical waste samples, and agricultural mixtures were dissolved in acetone and analyzed by gas chromatography− mass spectrometry (GC−MS) to determine the purity or the relative abundances of different isomers. Pure γ-HCH from pharmaceutical formulations was extracted by vortex mixing with DCM. The extraction procedure and analytical methods for GC−MS are described in detail in SI sections 2 and 3. Carbon Isotope Analyses. The carbon isotope ratios were expressed in the delta notation (δ13C) and reported in per mil (‰).34 Carbon EA−IRMS Analyses for Pure Crystalline Samples of HCH (Purity ≥ 95%). We determined carbon isotope ratios of pure HCHs by elemental analysis−isotope ratio mass spectrometry (EA−IRMS) using a two-point calibration against the Vienna Pee Dee Belemnite (V-PDB) scale, as previously described elsewhere.35 The detailed analytical method for EA− IRMS is described in SI section 4. Carbon GC−C−IRMS Analyses for HCH-Containing Mixtures (Purity < 95%). Compound-specific carbon isotope ratios of target HCH isomers in HCH-containing mixtures were determined by gas chromatography−combustion−isotope ratio mass spectrometry (GC−C−IRMS, SI section 5), as described elsewhere.17 Hydrogen Isotope Analyses. The hydrogen isotope ratios are expressed in the delta notation (δ2H) and reported in per mil (‰).34 Hydrogen EA−Cr/HTC−IRMS Analyses for Pure Samples of HCH (Purity ≥ 95%). Hydrogen isotope ratios of pure HCHs were determined using a novel chromium-based continuousflow conversion method in which the high-temperature conversion of heteroatom-containing compounds is combined with chromium reduction (Cr/HTC) to H2 in an elementalchromium-filled reactor coupled to an IRMS analyzer.18 The analytical method for EA−Cr/HTC−IRMS is described in detail in SI section 6. In the present study, we validated the method for the complete conversion and accurate determination of hydrogen isotope composition of HCHs, as complete combustion is crucial for an accurate determination of the δ2H values of polyhalogenated compounds.31 Complete conversion of HCH was evaluated through the analysis of byproduct formation during conversion by coupling an ion-trap mass spectrometer to the EA−IRMS system as described elsewhere18 (SI section 7). For that purpose, a continuous sequence including 36 HCH samples and various chlorinated and nonchlorinated standard materials was monitored. A two-point calibration against the standard mean ocean water (SMOW) scale was applied using international standards (GISP and VSMOW), and the accuracy was confirmed with the international reference material IAEA-CH7 (δ2H value of −100‰) and hexadecane standards USGS67, USGS68, and USGS69 with δ2H values of −166‰, −10‰, and +81‰, respectively. The standards were measured after each 10 samples in the analytical sequence.

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RESULTS AND DISCUSSION Characterization of Sample Collection. Our collection of 77 HCH samples represents production locations from at least 15 different countries worldwide covering a period of about 55 years from the 1960s until now. Information about sample type, origin, age, isomeric composition (Tables SI-1.1 and SI-2.1) and other specifications was summarized for a data bank, along with that on previously reported HCH samples (Table SI-3.1). For some analytical standards from suppliers or branched companies and for some agricultural and pharmaceutical formulations, information about production location is still missing. These samples were labeled as unknowns but were, nevertheless, included in the data bank to show the overall variability of isotope compositions. The isomeric compositions allow the samples to be divided into two groups: (i) chemically and isomerically pure samples containing only one HCH isomer with a purity of ≥95% and suitable for bulk isotope analyses by EA−IRMS and (ii) mixtures containing several HCH isomers and/or single isomers with a purity of 95% for HCHs (generally >98%), confirming that the conversion reaction was complete. The negligible amounts of HCl and other chlorinated byproducts observed by ion-trap MS analysis during the combustion process also serve as a confirmation of complete conversion. After optimization of the flow parameters and within the linearity range of the GC−Cr/HTC−IRMS method, the δ2H values of HCHs in mixtures could be determined with a precision of less than or equal to ±5‰ (SI section 8). Two similar reactors used for the high-temperature conversion of HCHs exhibited slightly different performances, but the correlations of δ2H values compared to EA−HTC−IRMS were similar. We determined the δ2H values of 42 pure HCH isomers with the validated EA−Cr/HTC−IRMS method (Table SI-1.2). Application of the same conversion principle for CSIA allowed 12 δ2H values of γ-HCH and 17 δ2H values of α-HCH to be obtained for extracts from formulations and isomeric mixtures by GC−Cr/HTC−IRMS (Table SI-2.2). Analyses of other samples were obstructed by low chromatographic resolution (especially peaks of β- and γ-HCH). Attempts were made to optimize the flow and temperature program to improve the peak separation. However, it was not possible to reach complete baseline separation because of a broad peak shape and peak tailing (see SI section 8). In addition, α-HCH was a main component with a relative abundance ≥80% in many mixtures and waste samples. Analysis of other HCH isomers of lower relative abundances (≤10%) would require the preparation of solutions with high summed concentrations of HCHs in which the concentration of α-HCH would exceed its solubility level (140 g/L in acetone). Therefore, analyses of δ2H values of β-, δ-, and γ-HCH isomers at lower relative abundances were not always possible, as the mixture could not be completely dissolved. D


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result in carbon isotope enrichment (Δδ13C) of 8‰ at a degradation level of 99.6% (for calculations, see SI section 11). This enrichment corresponds to the overall range of source carbon isotope signatures of γ-HCH observed in this study, demonstrating that isotope fractionation during biodegradation needs to be taken into account in any attempt to analyze sources of HCH. Thus, the most positive source carbon isotope ratio of the corresponding HCH isomer reported [e.g., −24.9‰ for γ-HCH (M14) and −22.7‰ for β-HCH (M10), Table SI-2.2] can serve as an assessment of HCH biodegradation. Taking into account an uncertainty level of 2‰ for considering isotope effects of physical processes and sampling,39 δ13C values of γ-HCH that are more positive than −22.9‰ would provide a strong indication for degradation processes. Moreover, the precision of carbon isotope measurements (less than or equal to ±0.5‰ and less than or equal to ±0.2‰ for 84% of the samples) together with the variability of δ13C values of HCH isomers within a range of about 13.2‰ can be used to distinguish different HCH sources. Chlorine Isotope Ratios. Similarly to the carbon isotope ratios, the majority of the δ37Cl values obtained for HCH fall in the relatively narrow range between −1.60‰ and +0.54‰, close to the expected chlorine isotope ratio of the raw material (0‰ for seawater chlorides and salts30,32). Two γ-HCH samples (P32 and P33, Tables SI-1.1 and SI-1.2) produced from a known source of chlorine gas (Aragonesas Industrias, Madrid, Spain) yield δ37Cl values of −1.46‰ and −1.55‰. These small variations can provide some indications about the regional source of chlorine or the production method. From various reports on mercury pollution, it is known that Aragonesas Industrias produced chlorine by the mercury-cell method, as did the majority of west European chlorineproducing plants before 1998.40 Thus, the slight depletion in 37 Cl for two other γ-HCH samples, produced in Europe and the United States between 1975 and 1989 (P2 with a δ37Cl value of −1.43‰ and M23 with a δ37Cl value of −1.60‰, respectively) (Figure 1 and Tables SI.1.1 and SI. 1.2) could result from this specific source of chlorine gas for the synthesis of HCH. On the other hand, a group of samples from Asian countries (P3 and P16 from India; P9, P10, and P29 from China; and P34 from Japan) (Figure 1 and Tables SI.1.1 and SI. 1.2) have δ37Cl values between −0.12‰ and −0.70‰, which could indicate another regional source or production method of chlorine. Together with the high precision of the analytical method applied (±0.11‰, 1σ, for the entire conversion procedure and DI-IRMS analyses23), these regional trends allow chlorine isotope ratios to be used as an additional descriptor for distinguishing between HCH sources with similar δ13C and δ2H values. Chlorine isotope fractionation upon degradation of HCH has not been thoroughly studied, but small variations in the source signal of about 2‰ might allow for the characterization of the biodegradation when the chlorine isotope fractionation factor is high enough, as was recently found for other chlorinated compounds.41,42 Hydrogen Isotope Ratios. Hydrogen isotope fractionation is often more pronounced than for other chemical elements because of the large mass difference between 2H and 1H. A larger variation in the δ2H values compared to the δ13C values was also reported before for HCH and other chlorinated pesticides,28 showing that greater hydrogen isotope fractionation can occur during HCH synthesis and purification processes. The range of δ2H values obtained for the majority of samples (group I) was between −102‰ and −52‰, which

Figure 2. Variability of δ13C and δ2H values for HCH sources. Blue diamonds represent γ-HCH; red squares, α-HCH; green triangles, βHCH; and purple circles, δ-HCH. Red dashed lines show the ranges of carbon13 and hydrogen15,16 isotope ratios of benzene from fossil stocks as a raw material for HCH production. Ovals highlight selected groups with significantly different isotope compositions.

To understand the possible reasons for specific stable isotope ratios, it is necessary to identify the isotope-sensitive critical steps of the synthesis and purification processes. During the synthesis of HCH by chlorine addition, the π-electron system of benzene is permanently broken, and new carbon−chlorine bonds are formed. Thus, the continuous-flow photochlorination process or the incomplete conversion of benzene in the batch reactor can lead to kinetic isotope effects for carbon and chlorine. In addition, various recrystallization steps, applied to separate γ-HCH from other HCH isomers, can also cause isotope effects for all three elements in HCH. We evaluate the possible reasons for the original isotope fingerprints of each of the distinct groups (groups II to V) in a separate section (Groups of Samples with Significantly Different Multielemental Isotope Compositions). To investigate the general trends in the isotope ratios, we first evaluate each element separately with respect to the production time and location. Carbon Isotope Ratios. The majority of carbon isotope ratios lie between −31‰ and −25‰, falling within the range of δ13C values of benzene from fossil stocks (between −33‰ and −19‰13). Because of the lack of isotope forensic studies on benzene from different locations worldwide, it is difficult to assign different isotope ratios of HCH produced in the past to the raw materials from which it was synthesized. The relatively narrow range of carbon isotope ratios of HCH indicates that only minimal carbon isotope fractionation occurs during the production process. This assumption is also supported by the identical range of carbon isotope ratios (between −33‰ and −26‰) of γ-HCH in untreated technical mixtures (t-HCH, isomeric content of γ-HCH of ≤12%) and mixtures enriched in γ-HCH (isomeric content between 12% and 70%) and very similar to the range for purified γ-HCH (from −31‰ to −25‰ for an isomeric content of ≥90%) (Figure SI-9 and SI section 10). Similar trends were found for the other HCH isomers. All isotope ratios reported here correspond to samples that can be considered as sources, not affected by transformation processes. Aerobic biodegradation of γ-HCH by Sphingobium indicum with a carbon isotope enrichment factor of −1.5‰ ± 0.1‰25 according to the Rayleigh equation model38 would E


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Interpretation of Isotopic Compositions with Respect to Production Time and Location. The ranges of carbon and chlorine isotope ratios did not change systematically during the entire observed production period (from the late 1960s until now) for all HCH isomers, suggesting that the source isotope compositions are uncertain indicators for the dating of production (Figure 3).

is comparable to the range of hydrogen isotope ratios in benzene (−97‰ to −28‰16). However, a number of samples with significantly more positive and negative δ2H values expanded the overall range of hydrogen isotope ratios from −233‰ to +1‰. Taking the precision of less than or equal to ±5‰ into account, the hydrogen isotope ratio can serve as an excellent descriptor for source tracking of HCH. Groups of Samples with Significantly Different Multielemental Isotope Compositions. The combination of the carbon, hydrogen, and chlorine isotope data allows five groups of HCH samples with significantly different multielemental stable isotope compositions to be identified: Group I is the main group and contains about 73% of all samples from different production times and locations. Group II (same production method, Figures 1 and 2) consists of two samples of purified isomers without insecticidal properties, α-HCH (P4) and β-HCH (P5), from the same modern supplier of analytical materials with δ13C values of −33.8‰ and −34.1‰, respectively (Tables SI-1.1 and SI-1.2). These HCH samples are more depleted in 13C than the lightest benzene source, with a δ13C value of −33.0‰. Additionally, these two samples are significantly depleted in 37Cl, with δ37Cl values of −4.68‰ and −6.69‰, respectively. The negative carbon and chlorine isotope ratios of these samples seem to result from specific synthetic and/or purification processes, applied to enrich nonactive isomers from technical mixtures or probably even from chemical waste after the isolation of γHCH. Group III (same benzene source, Figures 1 and 2) consists of two samples of purified δ-HCH from different modern suppliers of analytical materials (P14 and P22, Tables SI-1.1 and 1.2). These samples have δ13C values of −35.8‰ and −35.9‰ and δ2H values of −21‰ and −36‰, respectively. Relative to raw benzene, they are depleted in 13C and enriched in 2H. These specific δ13C and δ2H values along with δ37Cl values of 0.07‰ and 0.22‰, close to that of seawater-derived chlorine, might indicate a specific benzene source used for the production of these two δ-HCH samples. Group IV (fine chemicals, Figures 1 and 2) consists of four samples of purified analytical-grade γ-HCH (P10, P16, P29, and P34 in Figure 1 and Tables SI-1.1 and SI-1.2) produced in India, China, and Japan, as well as samples P18 and P37 from an unknown production location supplied by Sigma-Aldrich (Figure 2). All of these samples have average δ13C values but are slightly depleted in 2H. In addition, their chlorine isotope ratios are within the range between −0.27‰ and −0.7‰, typical for the Asian region (see the section Chlorine Isotope Ratios). These specific values might indicate a common strategy applied for the production of HCH in several plants located in Asian countries, and thus, they might serve as a regional marker for source investigation. Only one sample produced in the same region (P3, Tables SI-1.1 and SI-1.2) has an isotope fingerprint within the medium range of isotope ratios and does not fit in this group. Group V (pharmaceuticals, Figure 2) consists of three samples of purified γ-HCH extracted from pharmaceutical formulations (M5, M6, and M7, Tables SI-2.1 and SI-2.2). These are all pharmaceutical products analyzed within our study. They are significantly depleted in 2H, with δ2H values between −175‰ and −233‰. The sspecific isotopic compositions of these samples might result from multiple purification steps, needed to obtain the very high chemical purity of γ-HCH required for pharmaceutical applications.

Figure 3. δ13C, δ2H, and δ37Cl values of HCH isomers in pure samples and in the mixtures with known production times (total of 65 samples, Tables SI-1.1−SI-1.3 and SI-2.1−SI-2.3). Blue diamonds represent γHCH; red squares, α-HCH; green triangles, β-HCH; and purple circles, δ-HCH. Red dashed lines show the ranges of the carbon13 and hydrogen15,16 isotope ratios of benzene from fossil stocks as a raw material for HCH production, as well as the chlorine isotope ratio of seawater chlorine.20

However, there were variations in carbon isotope ratios ranges over the years that seem to be related to the number of factories dedicated to the production of HCHs at a certain period of time and, consequently, more probable variations within their production processes. We found a broader range of carbon isotope ratios for γ-HCH (δ13C values between −32.8‰ and −24.9‰) between the 1980s and the late 1990s, when HCH was produced in many factories around the world. During these years, attempts were made to optimize the production process to minimize waste isomer formation,9 for example, the optimization of the photochlorination reaction conditions or the replacement of UV irradiation by the application of different catalysts for the chlorination reaction. After the Stockholm Convention in 2009, only a few plants in the world have continued large-scale production of γ-HCH, probably applying similar technologies, which correlates to a very narrow range of recent carbon isotope ratios (between −28.3‰ and −25.8‰). Most of the hydrogen isotope ratios of γ-HCH produced after the year 2000 are more negative than the δ2H values of raw benzene (Figure 3), which is probably related to the stronger requirements on the purity of HCH for recently produced analytical materials and pharmaceuticals. We observed significantly more negative δ2H values for some pure γ-HCH samples from earlier production as well. For example, M1 produced in 1981 with a δ2H value of −149‰ and M7 produced in 1999 with a δ2H value of −233‰ indicate a stronger dependency of the hydrogen isotope ratio on the HCH purification procedure rather than the production time. Comparison of carbon and hydrogen isotope ratios from a number of recent sources (SI section 12 and Figure SI-11) reveals relatively high batch-to-batch similarities of stable F



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isotope composition, as well as possible exchange of raw and purified chemicals between subdivisions of the same company and, probably, between different producers and suppliers. In addition, comparative analysis of three γ-HCH samples produced at two German companies in the 1970s through the early 1980s (SI section 12 and Figure SI-12) confirms the higher dependency of the carbon isotope ratio on the synthesis of t-HCH and the higher dependency of the hydrogen isotope ratio on the γ-HCH isolation and purification processes. The carbon and hydrogen isotope ratios of HCH are largely independent of the production country, showing no clear regional trends (Figures SI-13 and SI-14). A possible reason for this unification might be the globalized market of the raw material (benzene) and different production methods applied at different companies within the same country. Implications for Isotope Forensic Studies. Tracking sources of HCHs remains an important environmental issue, and a better understanding of their transformation processes and sinks is essential for the accurate assessment of contaminated sites. The comprehensive data set on carbon, hydrogen, and chlorine isotope ratios of HCHs compiled in this work provides a baseline for multielemental stable isotope analysis for environmental forensic investigations. The relatively low analytical uncertainties of the novel methods for stable isotope analysis make it possible to provide an accurate and robust multielemental stable isotope fingerprint of HCHs. Therefore, it is possible to differentiate between two given samples, even when the vast majority of the samples from different geographical origins and production times fall within a group with moderate ranges of carbon, hydrogen, and chlorine isotope compositions. It was possible to identify some groups of samples with stable isotope fingerprints that differ significantly from the group with medium isotope signatures. The distinct stable isotope compositions of these samples can be related to specific purification processes applied in certain areas, such as Asian countries, or to specific types of products such as pharmaceuticals. These findings can be applied for tracking sources, transport, and degradation pathways of these samples in the environment at a global scale. In addition, the most positive δ2H, δ13C, and δ37Cl values reported here can serve as baselines for degradation studies on the regional scale, considering 2H-, 13C-, and 37Cl-enriched HCHs as evidence for the degradation processes occurring. Some analytical limitations still constrain the application of multielemental stable isotope fingerprinting to samples from contaminated field sites, samples containing low concentrations of HCHs, and samples containing complex mixtures of compounds. These are basically (i) the lack of a robust chlorine CSIA method for HCHs at low concentrations and (ii) the need for improvement of the low sensitivity and baseline separation of GC−Cr/HTC−IRMS for hydrogen isotope analysis. However, we have demonstrated that multielemental stable isotope fingerprinting for the source identification of HCHs of commercial producers, chemical stocks of former producers, and waste dumps is possible. We believe that further improvements of existing analytical methods for chlorine and hydrogen CSIA33,43 will allow routine multielemental stable isotope fingerprinting of HCHs in environmental samples with low concentrations.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.6b03039. Information about synthesis and purification of γ-HCH; detailed procedures and instrumentations for sample preparation, GC−MS, carbon EA− and GC−C−IRMS analyses, hydrogen EA− and GC−Cr/HTC−IRMS analyses, and chlorine offline DI-IRMS analyses; details and results of optimization and validation of the methods for hydrogen EA and GC−Cr/HTC−IRMS analyses; discussion of the correlation between the relative abundance of different HCH isomers and their isotope values; calculations of changes of carbon isotope ratios during biodegradation; and discussion of the distribution of isotope values according to origin (PDF) Descriptions and isotopic compositions of the samples (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49-3412351212; fax: +49-341451212; e-mail: hans. [email protected]. ORCID

Hans-Hermann Richnow: 0000-0002-6144-4129 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully and sincerely thank Falk Brattfisch, Ursula Günther, Matthias Gehre, Tetyana Gilevska, Liu Yaqing, Julian Renpenning, Steffen Kümmel, and Kristina L. Hitzfeld for analytical support and helpful discussions on the improvement of applied methods. We acknowledge Sascha Wagner for the preparation of quartz ampules needed for chlorine isotope analysis and Pedro Inostroza for assistance with graphical software. We offer sincere thanks to Thomas Krauss, Marilda Tedesco, Martin Forter, Tomás ̌ Ocelka, Rup Lal, Walter Vetter, Ralph Kretschmann, Alfred Jumar, Werner Kochmann, and Horst Ulrich for providing HCH samples and to Safdar Bashir and Yaqing Liu for their contributions at different stages of the preparation of the manuscript. We give special thanks to Jesús Fernández Cascan (Departamento de Medioambiente del Gobierno de Aragón) for his interest in our study and his effort to provide a wide collection of samples of HCHs form the former HCH production plant Inquinosa (Sabiñań igo, Huesca, Spain). M.B. thanks CNRS INSU for funding and supporting the Cl isotope laboratory and her salary (IPGP contribution 3805). M.B. and T.G. thank Gérard Bardoux for his help in the maintenance of the Cl stable isotope laboratory at IPGP. In addition, N.I. and S.H.-M. acknowledge funding from CSI:Environment Initial Training Network within Marie Skłodowska-Curie Actions. The research reported in this article was supported by the European Union under FP7-People-ITN2010 (Grant Agreement 264329). G


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