Natural Chlordecone Degradation Revealed by Numerous

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Characterization of Natural and Affected Environments

Natural chlordecone degradation revealed by numerous transformation products characterized in key French West Indies environmental compartments Marion Laure Chevallier, Oriane Della-Negra, Sébastien Chaussonnerie, Agnès Barbance, Delphine Muselet, Florian Lagarde, Ekaterina Darii, Edgardo Ugarte, Ewen Lescop, Nuria Fonknechten, Jean Weissenbach, Thierry Woignier, Jean-François Gallard, Stéphane Vuilleumier, Gwenaël Imfeld, Denis Le Paslier, and Pierre-Loïc Saaidi Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06305 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 15, 2019

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Environmental Science & Technology

ACS Paragon Plus Environment


chlordecone

1

Natural

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numerous transformation products characterized in

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key

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compartments

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Marion L. Chevalliera,1, Oriane Della-Negraa,1, Sébastien Chaussonneriea,2, Agnès Barbancea,2,

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Delphine Museleta,2, Florian Lagardea, Ekaterina Dariia, Edgardo Ugartea, Ewen Lescopb,

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Nuria Fonknechtena, Jean Weissenbacha, Thierry Woignierc, Jean-François Gallardb, Stéphane

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Vuilleumierd, Gwenaël Imfelde, Denis Le Pasliera,*, Pierre-Loïc Saaidia,*.

French

degradation

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West

Indies

revealed

by

environmental

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a

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Université Paris-Saclay, Evry, France.

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b

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Terrasse 91198 Gif-sur-Yvette Cedex, France.

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c

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Marseille Université, CNRS, IRD, Avignon Université and IRD, UMR IMBE, Campus Agro

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Environnemental Caraïbes B. P. 214 Petit Morne, 97235, Le Lamentin, Martinique, France.

Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry,

Institut de Chimie des Substances Naturelles. CNRS - UPR 2301 Bâtiment 27, 1 avenue de la

Institut Méditerranéen de Biodiversité et d'Ecologie marine et continentale (IMBE), Aix


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d

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7156 CNRS, 4 allée Konrad Roentgen, 67000 Strasbourg, France.

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e

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UMR 7517 CNRS/EOST, 1 Rue Blessig, 67084 Strasbourg Cedex, France.

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1

M.C. and O.D.N. contributed equally

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2

S.C., A.B. and D.M. also contributed equally

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*

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[email protected] or [email protected].

Génétique Moléculaire, Génomique, Microbiologie (GMGM), Université de Strasbourg, UMR

Laboratory of Hydrology and Geochemistry of Strasbourg (LHyGeS), Université de Strasbourg,

To whom correspondence may be addressed: Pierre-Loïc Saaidi, or Denis Le Paslier, E-Mail:

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ABSTRACT

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Production and use of the insecticide chlordecone has caused long-term environmental pollution

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in the James River area (US) and the French West Indies (FWI) that has resulted in acute human-

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health problems and a social crisis. High levels of chlordecone in FWI soils, even after its ban

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decades ago, and the absence of detection of transformation products (TPs), have suggested that

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chlordecone is virtually non-biodegradable in the environment. Here, we investigated laboratory

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biodegradation, consisting of bacterial liquid cultures and microcosms inoculated with FWI soils,

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using a dual non-targeted GC-MS and LC-HRMS approach. In addition to previously reported,

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partly characterized hydrochlordecones and polychloroindenes (families A and B), we

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discovered 14 new chlordecone TPs, assigned to four families (B, C, D, E). Organic synthesis

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and NMR analyses allowed us to provide the complete structural elucidation of 19 TPs.

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Members of TP families A, B, C and E were detected in soil, sediment, and water samples from

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Martinique, and include 17 TPs not initially found in commercial chlordecone formulations.

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2,4,5,6,7-pentachloroindene was the most prominent TP, with levels similar to those of

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chlordecone. Overall, our results clearly show that chlordecone pollution extends beyond the

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parent chlordecone molecule and includes a considerable number of previously undetected TPs.

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Structural diversity of the identified TPs illustrates the complexity of chlordecone degradation in

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the environment, and raises the possibility of extensive worldwide pollution of soil and aquatic

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ecosystems by chlordecone TPs.


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KEYWORDS. Chlordecone │ kepone │ persistent organic pollutants │ pollution │

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biodegradation │ structure elucidation│ transformation products


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INTRODUCTION

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Organochlorine (OC) compounds have been used extensively worldwide for decades, for insect

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control in agriculture. Their use has gradually been prohibited since the 1970s because of

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biological biomagnification, high toxicity, and their long persistence in the environment. Many

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OC compounds synthesized from hexachlorocyclopentadiene1 belong to the Stockholm

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convention list of persistent organic pollutants (POPs), which includes aldrin, dieldrin, endrin,

57

chlordan, heptachlor, mirex, chlordecone, and endosulfans. Among them, the insecticides

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chlordecone and mirex (which has also been commonly used as a fire retardant) represent a

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particular class of OC compounds obtained from hexachlorocyclopentadiene dimerization1, 2.

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They share several characteristics such as low molecular orbital energy, high hydrophobicity,

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and low Kow3. Chlordecone is also known to be a transformation product (TP) of mirex4 and

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kelevan, another insecticide mainly used in Europe5. Their highly stable perchlorinated

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bishomocubane structure renders such OC compounds extremely recalcitrant towards

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environmental conditions. Chlordecone formulations from the US (such as Kepone) and Brazil

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(such as Curlone) have mainly been used in the Caribbean area, Central America, and Africa6,

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whereas the use of mirex has been reported in North America7, 8, Latin America, Europe6, and

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more recently China9. Because of its direct use or in situ formation, chlordecone is thus

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essentially present throughout the world, and has even been detected in the coral reefs of French

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Polynesia10. We thus studied chlordecone as a relevant and challenging pollutant of the

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hexachlorocyclopentadiene-based OC class.

71

To date, chlordecone has been associated with two major environmental disasters: (1)

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environmental contamination near the Hopewell chlordecone production plant (U.S.) in 1975,

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resulting in acute exposure of workers and massive pollution of the James River, extending over


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100 miles, that lasted for decades11. On a larger scale, extensive use of chlordecone in French

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West Indies (FWI) banana plantations from 1972 to 1993 has resulted in long-term pollution of

76

environmental compartments and the local food chain (soil, water resources, farmed animals, and

77

fish)6, 12, 13. Chronic exposure to chlordecone has also resulted in human health problems14-26 and

78

subsequent socio-economic problems for the FWI and James River area27-30.

79

Following its application, chlordecone is absorbed by soil and sediment particles, especially soils

80

and sediments of FWI with high organic content. The commonly accepted paradigm of the

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persistence of chlordecone for decades, and even centuries, in the FWI, based on a leaching

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model31, was recently comforted by two studies that suggested only marginal degradation in

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tropical soils, if any32,

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environmental monitoring of the parent molecule, with only limited efforts dedicated to detecting

85

TPs potentially formed in situ and assessing their impact. In the case of chlordecone, several

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laboratory studies on bacterial degradation of chlordecone suggested formation of two main TP

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families, as shown by GC-MS analysis: hydrochlordecones arising from reductive dechlorination

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(family A), and polychloroindenes formed after ring-opening and elimination steps (family B)34-

89

37

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structures of these TPs has been completely elucidated, i.e., all hydrogen and chlorine atoms

91

have not been correctly positioned on core structures. In addition, the Archaeon Methanosarcina

92

thermophila has been shown to convert chlordecone into unknown polar and nonpolar products,

93

based on silica gel thin-layer chromatography analysis39. A definitive assignment of these TPs to

94

the hydrochlordecone and polychloroindene TP families could not be obtained. Despite extensive

95

analytical studies on chlordecone contamination in the FWI6, the only chlordecone derivatives

96

detected in environmental samples so far, i.e. chlordecol and 8-monohydrochlordecone, turned

33

. Traditionally, POP contamination is assessed and followed by

. With the exception of 8-monohydrochlordecone and 2,8-dihydrochlordecone38, none of the


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out to be contaminants of commercial formulations of chlordecone. Moreover, the existence of

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other TPs cannot be excluded at present, as only targeted analyses have been applied. Although

99

chlordecol and 8-monochlordecone are the only commercially available chlordecone derivatives,

100

other TPs from families A and B have been reported following exposure of chlordecone to

101

specific chemical degradation protocols. For example, UV-irradiation can lead to the production

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of 8-mono- and 2,8-di-hydrochlordecones in very low yields38, and reductive degradation of

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chlordecone in the presence of vitamin B12 and titanium citrate gives rise to TPs of both families

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A and B37. Importantly, all other successful protocols for chemical degradation of chlordecone

105

have required a reducing agent, either alone40 or in combination with vitamin B1237, 39, 41, 42.

106

Here, we addressed the paradigm of the non-biodegradability of chlordecone in the FWI

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environment through the prism of its TPs: (i) by extending the currently known library of

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chlordecone TPs36, relying on anoxic microbial degradation and the use of untargeted GC-MS

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and LC-HRMS; (ii) through the development of chemical degradation protocols for synthesizing

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and purifying TPs at the milligram scale; (iii) by structural elucidation of isolated TPs using

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NMR spectroscopy combined with chemical derivatization; and (iv) by use of the newly

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elucidated TPs as analytical standards for TP detection in FWI soil, water, and sediment samples.


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MATERIALS AND METHODS

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Chemicals and Analytics

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All used chemicals and analytical and purification methods are described in detail in the

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Supporting Information (SI) (Supporting Methods).

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Analytical methods

118

GC-MS analysis

119

GC-MS analyses were carried out using a Thermo Fisher Focus GC coupled to a single-

120

quadrupole mass spectrometer (Thermo Fisher DSQ II). The instrument was equipped with a

121

non-polar 30 m × 0.25 mm × 0.25 µm DB-5MS column (Agilent J&W) and a split/splitless

122

injector. Ionization conditions and GC program were described elsewhere36,

123

chromatographic methods are provided in SI.

124

LC-HRMS analysis

125

LC-HRMS analyses were carried out using a Dionex Ultimate 3000 LC system coupled to an

126

LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) fitted with a heated

127

electrospray ionization source (HESI) operating in negative ionization mode. Voltage

128

optimization was described elsewhere36. Chromatographic separation was achieved using a

129

Thermo Fisher Syncronis™ C18 column (50 mm length, 2.1 mm inner diameter, 1.7 µm particle

130

size). Detailed chromatographic methods are provided in SI.

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Anoxic microbial incubations

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. Detailed


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Bacterial cultures were incubated at 24°C in a glove box (Unilab mBraun) under anaerobic

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conditions in an N2/H2 (98/2; V/V) atmosphere in modified MM+ medium36 containing Na2S

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(0.4 g/L) as a reducing agent.

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Bacterial degradation of chlordecone in the presence of consortium 86 and Citrobacter sp. 86

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Modifications of the previously described MM+ medium36 were as follows: MgCl2 and NH4Cl

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were replaced by MgSO4 and (NH4)2SO4, respectively. NaCl, CaCl2, KCl were left out. Two

138

liters of freshly prepared medium were inoculated with an active consortium 86 culture (1/100

139

v/v). After 8 h of incubation, three glass bottles were filled with 650 ml growing bacterial

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culture, and another with medium alone as a negative control. Chlordecone (162.5 µl of a

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solution of 200 mg chlordecone in 1 ml dimethylformamide) was added to a final chlordecone

142

concentration of 50 mg/L in each bottle. The same procedure was carried out with Citrobacter

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sp. 86. Cultures were incubated and monitored for 250 days by GC-FID, GC-MS, LC-HRMS,

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and chloride analysis.

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Chlordecone biodegradation in soil/liquid microcosms from Guadeloupe Island

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Soil microcosms were prepared from 0.5 g dry weight (dw) andosol or 0.5 g dw nitisol sampled

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from Guadeloupe Island. Each sample was inoculated under an oxygen-free nitrogen atmosphere

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containing 5% H2, at 25 °C, into 12 mL M9 mineral medium or M9 mineral medium

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supplemented with vitamin B12 (2 mg/L)43. Duplicate samples were taken over 36 months, using

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a sacrificial approach. A microcosm pair (duplicate samples) was retrieved from each series and

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analysed at t = 0, t = 10 months, and t = 36 months. Two negative-control series were conducted

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for each series (again in duplicate, for t = 0 and t = 36 months) and consisted of (a) aerobically

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incubated soil samples and (b) irradiated soil samples (30 ± 1.5 kGy).


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Duplicate samples from t = 0, t = 10, and t = 36 months were first basified to pH 12 with NaOH

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(1 M) and extracted with pentane (6 x 15 mL) after vortexing and decanting. Aqueous phases

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were then acidified to pH 1 with HCl (1 M) and extracted with CH2Cl2 (12 x 15 mL) after

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vortexing and decanting. Organic layers were pooled, concentrated in vacuo, and analyzed in

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duplicate by GC-MS and LC-HRMS (Figure S3, Tables S2-S3).

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Chemical access to chlordecone TPs

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All chemical chlordecone transformation experiments were monitored by GC-MS and LC-

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HRMS.

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Preparation of TP A1 (10-monohydrochlordecone)

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Sodium sulfide (2.2 g, 2.8 10-2 mol, 140 eq.) and vitamin B12 (40 mg, 2.9 10-5 mol, 0.15 eq.) were

164

added to a solution of chlordecone (100 mg, 2.0 10-4 mol, 1 eq.) in degassed water (300 mL).

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The reaction was carried out under an N2 atmosphere at room temperature (rt) for 30 h, quenched

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with HCl (6 M) to pH 4.0, and degassed with N2 for 1 h to evacuate hydrogen sulfide. The

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aqueous reaction mixture was extracted with DCM (3 x 200 mL) and the combined organic

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phases concentrated in vacuo, resulting in a brown viscous residue. A first purification step was

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performed on the crude residue using a Combi Flash® Companion® Elution column at a flow

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rate of 40 mL/min using heptane as solvent A and a mixture of DCM/(CH3)2CO (1:1; v/v) as

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solvent B. Elution started with 0% B for 7 min, followed by a linear gradient, reaching 50% B

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within 5 min, a second linear gradient, reaching 100% B within 3 min, and further elution for 15

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min with 100% B. Fractions containing A1 (from 8 to 29 min) were pooled and concentrated

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under reduced pressure. A second purification step was performed using a preparative HPLC

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system. Isocratic elution using tetrahydrofuran/MeCN/(NH4)2CO3 buffer (10 mM, pH 9.5)

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(29:29:42; v/v/v) was applied at a flow rate of 20 mL/min. Fractions containing A1 (retention


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time of 9 min) were pooled, acidified to pH 3 with HCl (6 M), extracted three times with DCM,

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and concentrated under reduced pressure to give the title compound A1 (46.4 mg; 9.8 10-5 mol;

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50%) as a white solid. All NMR, GC-MS, and LC-HRMS analyses for A1 are provided in

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Figures S17, S26, and S30-S31.

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Preparation of TPs B1, B2 and B3-B4 (2,4,5,6,7-pentachloroindene, 4,5,6,7-tetrachloroindene,

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2,4,6,7-tetrachloroindene, 2,4,5,7-tetrachloroindene, respectively)

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Titanium(III) citrate (50 mL, 3.3 10-3 mol, 8.4 eq.) basified to pH 12.7 with NaOH (3 M) was

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added to a solution of chlordecone (200 mg, 3.9 10-4 mol, 1 eq.) and vitamin B12 (60 mg, 5.8 10-5

185

mol, 0.15 eq.) in degassed H2O/MeOH 64:36 (250 mL). The reaction mixture was stirred under

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N2 at room temperature for 80 min and quenched by exposure to O2. Extraction with pentane (5 x

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250 mL), followed by concentration under reduced pressure, gave rise to a white crude solid. TPs

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B1, B2, and B3-B4 were purified by preparative HPLC. Isocratic elution (MeCN/H2O 7:3; v/v)

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was applied at a flow rate of 25 mL/min. Fractions containing B1 (retention time of 42 min), B2

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(retention time of 28 min), and B3-B4 (retention time of 32 min) were pooled separately,

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extracted three times with pentane, and concentrated under reduced pressure. Each compound

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was then purified by PLC (Preparative Layer Chromatography; Merck, PLC Silica gel, 1 mm,

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F254, 20 x 20) (cyclohexane/EtOAc 9:1); the B1, B2, and B3-B4 retardation factors were 0.78,

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0.68, and 0.88, respectively. B1 (32.6 mg; 1.2 10-4 mol; 30%), B2 (3.0 mg; 1.2 10-5 mol; 3%),

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and B3/B4 (4.1 mg; 1.3 10-5 mol; 4%) were obtained as white solids. All NMR, GC-MS, and

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LC-HRMS analyses for B1, B2, and B3-B4 are provided in Figures S19-S20, S26, and S32-S48.

197

Preparation of 13C-enriched B1 is described in SI (Supporting Methods). Preparation of TPs C1-6

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and D1-4 is described in SI (Supporting Methods). All NMR, GC-MS, and LC-HRMS analyses

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are provided in Figures S22-S23, S26-S28, and S49-S69.


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Analyses of FWI environmental samples

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Field sites and soil sampling

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Samples were collected on Martinique Island. Soil (andosol, nitisol, and ferralsol) from the

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vicinity of the “Montagne Pelée” volcano and bed sediments from Galion bay were sampled

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from the 0–30 cm layer and conserved in a glass box. River and mangrove water samples were

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collected from 0–30 cm below the water surface in glass bottles. Samples were stored in the dark

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at 4°C until chemical extraction.

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Chemical extraction procedure

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Each sample was processed in duplicate. For soils and sediments (4 g), 15 mL of milliQ water

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was added, followed by acidification to pH 1 with HCl (1 M) and vortexing. After decanting, the

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supernatant was extracted with DCM (12 x 15 mL) and the pellet washed twice with DCM (15

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mL). For river and mangrove water, 0.75 L water sample was acidified to pH 1 with HCl (1 M)

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and extracted with DCM (12 x 350 mL). Organic layers were pooled, concentrated in vacuo, and

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analyzed in duplicate injections by GC-MS (in hexane/acetone 85:15) and LC-HRMS (in 10 mM

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NH4OAc buffer/MeCN 4:1) (SI, Supporting Methods). Soil samples from Martinique taken at

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locations known not to be contaminated with chlordecone (Nitisol 926) were used as negative

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controls, and were prepared and treated as mentioned above.

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RESULTS AND DISCUSSION

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Search for new TPs formed during microbial degradation of chlordecone

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To date, two families of chlordecone TPs have been identified during bacterial degradation of

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chlordecone34-37. Based on GC-MS analysis, the detected TPs were assigned

to


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hydrochlordecones (family A), with A1 as a monohydrochlordecone, and polychloroindenes

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(family B), with B1 (pentachloroindene) as main TP, and B2, B3 (tetrachloroindenes) as minor

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TPs36. Here, anoxic bacterial degradations of chlordecone in the presence of consortium 86 and

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Citrobacter sp. 8636 were replicated and analyzed using a set of complementary techniques

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(chloride titration, LC-HRMS, GC-FID and GC-MS) to search for new TPs of chlordecone

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Chloride concentration increased by 18.8 ± 0.9 mg/L (i.e., 5.5 ± 0.3 Cl atoms per chlordecone

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molecule) in consortium 86 liquid cultures, and by 19.7 ± 1.7 mg/L (i.e., 5.8 ± 0.5 Cl atoms per

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chlordecone molecule) in Citrobacter sp. 86 liquid cultures after 250 days (Figure S15),

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suggesting chlordecone dechlorination, which was confirmed here by both GC-MS and LC-

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HRMS (Figure 1, Figures S13 and S15).

232

Although previous GC-MS analyses suggested the predominance of TP B1 (C9Cl5H3) and, to a

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minor extent, the presence of A1 (C10Cl9H3O2)36, chloride release clearly suggested the

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formation of other so far undetected chlordecone TPs containing four (or less) chlorine atoms.

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GC-FID monitoring also confirmed the presence of volatile TPs A1 and B1 in all experiments

236

(Figure S14). In addition, untargeted LC-HRMS analysis revealed the formation of four

237

previously undetected polar chlorinated compounds (Ci, i = 1, 2, 3, 4) of generic formula C10Cl4-

238

nO2H4+n

239

content matched the observed chloride release in these experiments (Figure 1 and Figures S15,

240

S27-S28).

(n = 0, compounds C1 and C2; n = 1, compounds C3 and C4). Their low chlorine


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241 242

Figure 1. TP formation during bacterial chlordecone (CLD) degradation by Citrobacter sp. 86,

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monitored by (A) GC-MS full scan analysis (CLD (green), A1 (blue), B1 (pink) and B3 (light

244

pink)). (B) LC-HRMS analysis corresponding to extract ion chromatograms for quasimolecular

245

ion [M-H]- of m/z = 506.6797; 472.7187; 296.8852; 260.9271 (CLD (green), A1 (blue), C1

246

(purple), C2 (purple), C3 (magenta) and C4 (magenta), respectively).

247

In parallel, laboratory microcosms with contaminated FWI soils (typically containing between

248

0.1 and 30 mg chlordecone/kg of dry soil) were incubated in the dark for 36 months at room

249

temperature under a N2/H2 atmosphere, to evaluate the capacity of native FWI soil microbiota to

250

degrade residual chlordecone (Figure S3). For each condition (soil/liquid medium), two

251

replicates were collected over time by a sacrificial approach, to evaluate the produced

252

chlordecone TPs using the same analytical protocols as for liquid cultures.

253

GC-FID turned out to be inadequate due to high background from soil samples and low

254

chlordecone concentration. Untargeted GC-MS analyses (Table S2) demonstrated predominance

255

of TP B1, sporadic detection of A1, and the presence of B2 and B3 previously reported for

256

bacterial degradation of chlordecone36. In addition, a number of other chlorinated compounds

257

were detected: B0 (19.10 min), B5 (10.00 min), D1 (18.78 min), D2 (18.81 min), D3 (16.54

258

min), D4 (16.57 min), E1 (19.85 min), E2 (19.88 min), E3 (17.60 min and E4 (17.63 min). LC-


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HRMS analyses (Table S3), demonstrated the presence of Ci (i = 1, 2, 3, 4), already observed

260

during degradation of chlordecone in presence of consortium 86 and Citrobacter sp. 86.

261

Chlordecol, a known chlordecone contaminant in commercial formulations and present in FWI

262

soils at low levels44, was also found in all samples.

263

Mass spectrometric analysis of newly detected chlorinated compounds

264

The combined approach of GC-MS and LC-HRMS from in-source fragmentation chosen in this

265

study was sufficient to classify the newly detected chlorinated compounds into four distinct

266

families (the known polychloroindene family B, and three new families C, D, and E), and to

267

postulate a generic core structure for each of the families C, D and E newly identified in this

268

work (Figure 2).

269

Basing on GC-MS data, we first assigned compounds B0 and B5 to hexachloroindene and

270

trichloroindene, respectively. Indeed, their mass spectra contained ion series analogous to those

271

previously reported for pentachloroindene B1 and tetrachloroindenes B2 and B336 i.e. [C9Cl6-

272

nH2+n]

273

and B3) and n = 3 (B5) (Figures S18-S21).

274

The other chlorinated compounds detected in GC-MS were grouped into two new families (D

275

and E), with respective generic formulae of C11Cl4-nO2H6+n (n = 0, 1) and C12Cl4-nO2H8+n (n = 0,

276

1). The presence of ions [C10Cl4-nH3+nO2]+, [C10Cl4-nH3+nO]+, [C10Cl4-nH2+nO]+·, [C9Cl4-nH3+n]+· (n

277

= 0, 1) in the mass spectra of D1-D2 (n = 0) and D3-D4 (n = 1), displaying the same

278

chromatographic shape and probably corresponding to in-source losses of CH3·, CH3O·, CH4O

279

and C2H3O2· , suggested the presence of a methyl ester moiety in D1-D2 and D3-D4 (Figures S22

280

and S23). Similarly, for E1-E2 and E3-E4, presumed in-source losses of C2H4, C2H5O·, C2H6O

+·

, [C9Cl5-nH2+n] +, [C9Cl4-nH2+n] +·, [C9Cl4-nH1+n] + with n = 0 (B0), n = 1 (B1), n = 2 (B2


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and C3H5O2· were indicative of the presence of an ethyl ester moiety in E1-E2 and E3-E4

282

(Figures S24 and S25). In addition, the series of in-source fragment ions (C9ClxHy+/ C9ClxHy+·, x

283

= 6, 5, 4, 3, 2; y = 4, 3, 2, 1) were common to compounds Bi (i = 0, 1, 2, 3, 5), Dj (j = 1, 2, 3, 4),

284

and Ek (k = 1, 2, 3, 4). This suggested a shared polychloroindene aromatic ring, with methyl- and

285

ethyl-polychloroindenecarboxylate structures for families D and E, respectively (Figure 2).

286

Finally, the structure of chlorinated compounds Ci (i = 1, 2, 3, 4) only detected using LC-HRMS

287

was investigated in more detail. The ions [C9Cl4-nH3+n]- (n = 0, 1) observed in the negative mass

288

spectra of C1-C2 (n = 0) and C3-C4 (n = 1), displayed the same chromatographic shape in the

289

LC-HRMS run as the quasi-molecular ions [C10Cl4-nO2H3+n]- (n = 0, 1) (Figure S27 and S28).

290

Observed fragment masses correspond to the loss of CO2, likely indicating in-source

291

decarboxylation of C1-C2 and C3-C4. This observation, associated with similar UV-visible

292

absorption

293

polychloroindenecarboxylic acid structure for the new family C of chlorinated compounds

294

(Figure 2).

295

Chemical reductive degradation of chlordecone

296

We then investigated reductive degradation of chlordecone by chemical treatment in order to (i)

297

demonstrate the possibility of chlordecone to be transformed into the 14 newly detected

298

chlorinated compounds, (ii) complete the structural elucidation of chlordecone TPs using NMR

299

technique, and (iii) confirm the proposed generic structures for families C, D and E.

300

Chlordecone was first subjected to a selected set of 21 different chemical treatments, based

301

mainly on literature protocols37, 39-42, 45. This included treatment with a reducing agent alone, or

302

in the presence of a metal complex (Table S1). Dual GC-MS and LC-HRMS monitoring of

to

reported

indene

core-ring

profiles41

(Figure

S2),

suggested

a


16


Page 18 of 33

303

chemical reactions was applied to select the most suitable conditions, which were then further

304

refined for milligram-scale recovery of the major detected chlordecone TPs. Three conditions

305

(Table S1, entries a1, a4, and a6) expected to selectively produce polychloroindenes41, 42 resulted

306

in the formation of chlorinated compounds of families B, C, and D or E depending on the used

307

alcohol co-solvent (Figure S7). Titanium citrate and zero-valent iron without vitamin B12, in

308

contrast, oriented chlordecone degradation towards hydrochlordecone formation (Table S1,

309

entries e5 and e7). Polychloroindenes and chlorinated compounds of family C were formed

310

concomitantly at the highest levels upon addition of vitamin B12 in the presence of titanium

311

citrate and zero-valent iron (Figure 2 and Table S1, entries a5, a7).

312

Further, a combination of sodium sulfide and vitamin B12 specifically led to the production of

313

monohydrochlordecone A1 (up to a 50% yield after purification). From the same series of

314

experiments, compounds B1, B2, and B3 were isolated in yields of up to 30%, 3%, and 4%,

315

respectively. Using zero-valent iron and vitamin B12 (Table S1, entry a5), compounds C1-C2

316

(5% yield), C3-C4 (3% yield), and C5-C6 (traces) were obtained as non-separable pairs of

317

isomers. Indeed, when a single Ci (i = 1, 2, 3, 4) compound was isolated using preparative

318

HPLC, partial interconversion to its isomer occurred upon evaporation, demonstrating an

319

equilibrium between isomer pairs. Finally, supplementation of the reaction mixtures producing B

320

and C TPs with either methanol or ethanol led to the production of either D or E family

321

compounds. Purification of D1-D2 and D3-D4 isomeric pairs was achieved in 5% and 3% yield

322

respectively.


17

Page 19 of 33


323 324

Figure 2. Chlordecone TPs and their conditions of formation. (A) Chlordecone, chlordecol and

325

generic structures of the five TP families, and (B) TP profiles and associated release of chlorine

326

atoms in microbial or chemical treatment of chlordecone. 8-monohydrochlordecone was

327

assigned to A2. A3 and A4 correspond to dihydrochlordecones, and A5 to a trihydrochlordecone.

328

Complete elucidation of A1 structure


18


Page 20 of 33

329

Monohydrochlordecone A1, also formed after In Situ Chemical Reduction Daramend® treatment

330

of a FWI field46, was previously identified as either 9- or 10-monohydrochlordecone, based on

331

GC-MS and LC-MS fragmentations40. Replacement of a chlorine atom by a hydrogen atom in

332

chlordecone led to four different regioisomers, i.e., 6-, 8-, 9-, or 10-monohydrochlordecone

333

(Figure S5). Here, we unequivocally identified TP A1 as 10-monohydrochlordecone. Indeed, the

334

six distinct signals detected in its

335

symmetry which is only compatible with this specific regioisomer.

336

Complete elucidation of B1 structure

337

In our previous study, chemical derivatization allowed us to identify an indene aromatic ring in

338

TP B137. Here, we took advantage of the purified B1 to elucidate its structure using NMR. 1H

339

and COSY spectra (Figures S32, S34) indicated an allylic domain at  3.7 ppm (d, J = 1.4 Hz, 2

340

H), coupled with an aromatic proton at  6.87 ppm (t, J = 1.4 Hz, 1 H). Neither the 1.4-Hertz

341

value, compatible with a coupling constant of 3J- and 4J-type in the case of indene (Figure S29),

342

nor additional 1D- and 2D- NMR experiments (Figures S32-S36) allowed us to unequivocally

343

assign the position of the aromatic proton. Then, a

344

B1 synthesized from commercially available

345

every carbon atom to its direct carbon neighbors, and thereby to unequivocally assign B1 to

346

2,4,5,6,7-pentachloro-1H-indene by way of a HSQC experiment (Figure S35).

347

Complete elucidation of C1-C2 structures

348

Hydrogenation of the isolated C1-C2 mixture yielded, among other products, commercially

349

available 4-carboxyindane (Figure 3A and Figure S8). Taking into consideration the known

350

indene isomerization equilibrium47, we assigned C1 and C2 as tetrachloroindene-4-carboxylic

351

acid and tetrachloroindene-7-carboxylic acid, respectively. Comparison of 1H and

13

C NMR spectrum require a conserved vertical plane of

13

13

C-13C COSY experiment on

13

C-enriched

C8-chlordecone (Figure S38) allowed us to link

13

C spectra


19

Page 21 of 33


352

highlighted the similarity between TP B1 and C1-C2 (Figure 4A, and Figures S32, S49), and all

353

protons of C1-C2 were unequivocally positioned on the indene five-membered ring.

354

Interestingly, the HMBC NMR experiment of C1-C2 showed only one cross-correlation peak

355

between the most deshielded allylic protons and one of the two carboxylate carbon atoms (Figure

356

S53). We thus assigned this set of 1H and

357

carboxylic acid, i.e. C2, and the other set to 2,5,6,7-tetrachloro-1H-indene-4-carboxylic acid, i.e.

358

C1.

13

C signals to 2,4,5,6-tetrachloro-1H-indene-7-

359 360

Figure 3. Chemical derivatization of Ci (i = 1,2,3,4). (A) Formation of commercial standard 4-

361

carboxyindane from both C1-C2 and C3-C4 (LC-HRMS detection) (see Figure S8), (B)

362

Formation of D1-D2 and E1-E2 from C1-C2 (GC-MS detection) (Figure S6), and (C) Formation

363

of D3-D4 and E3-E4 from C3-C4 (GC-MS detection) (Figure S6).

364

Complete elucidation of B3-B4 structures

365

Tetrachloroindene B3 appeared as a single compound in GC-MS, but its 1H NMR spectrum

366

showed two similar but distinct sets of signals (Figure 4C). This suggested two regioisomers,

367

which were arbitrarily called B3 and B4. By analogy with 2,4,5,6,7-pentachloroindene B1, the


20


Page 22 of 33

368

methylene groups and the less-deshielded aromatic protons of B3-B4 were placed on carbons 1

369

and 3 (Figure 4A, C). Additional coupling of 0.6-0.7 Hz, observed only for B3 between protons

370

from carbon 1 and the most deshielded aromatic proton, was compatible with either a 2,4,5,7- or

371

a 2,4,5,6-tetrachloro substitution pattern. Of note, indene isomerization formally transferred

372

substituents from carbon 4 to carbon 7 and from carbon 5 to carbon 6. HMBC and HSQC

373

experiments indicated that both B3 and B4 featured a chlorine substituent at carbon position 4

374

(Figures S47-S48). We thus identified B3 and B4 as 2,4,6,7-tetrachloroindene and 2,4,5,7-

375

tetrachloroindene, respectively.

376

Complete elucidation of C3-C4 structures

377

Formation of 4-carboxyindane through hydrogenation of C3-C4 validated trichloroindene-4-

378

carboxylic acid and trichloroindene-7-carboxylic acid as the reactants (Figure 3A). This was

379

supported by the NMR spectra of B3-B4, which were highly similar to those of C3-C4 (Figure

380

4C). A strong HMBC cross-correlation peak between the less-deshielded protons and the

381

carboxyl carbons of C3 and C4 confirmed the structures of 2,5,7-trichloro-1H-indene-4-

382

carboxylic acid and 2,4,6-trichloro-1H-indene-7-carboxylic acid for C3 and C4, respectively

383

(Figure S53).

384

Complete elucidation of B2, C5-C6, D1-D2, D3-D4, E1-E2, and E3-E4 structures

385

Compounds B2 and C5-C6 showed the same 1H NMR pattern, allowing us to distinguish them

386

from B1, C1-C2, B3-B4, and C3-C4 (Figure 4). All 1H NMR signals were assigned to the five-

387

membered ring of the indene core. B2, C5, and C6 were thus identified as 4,5,6,7-tetrachloro-

388

1H-indene, 5,6,7-trichloro-1H-indene-4-carboxylic acid,

389

carboxylic acid, respectively. Finally, chemical derivatization of Ci (i = 1, 2, 3, 4) (Figure 3B-C)

390

proved that Dj (j = 1, 2 ,3 ,4) and Ek (k = 1, 2, 3, 4) were simply methylated and ethylated forms

and 4,5,6-trichloro-1H-indene-7-


21

Page 23 of 33


391

of the Ci (i = 1, 2, 3, 4) carboxylic acids (Figure 3B-C), respectively. NMR data collected for Dj

392

(j = 1, 2 ,3 ,4) comforted these conclusions (see detailed NMR interpretation in the SI

393

(Supporting text)).

394 395

Figure 4. 1H NMR spectra regions of selected indene-based TPs (600 MHz). Data were recorded

396

in (CD3)2CO excepted for B1, which was recorded in CDCl3.

397

Discovery and significance of new TPs of chlordecone


22


Page 24 of 33

398

Investigation of the diversity of TPs formed during chemical reductive degradation of

399

chlordecone demonstrates that the 14 new chlorinated compounds previously identified during

400

microbial experiments (B0 and B5; Ci, i = 1, 2, 3, 4; Dj, j = 1, 2, 3, 4; Ek, k = 1, 2, 3, 4) clearly

401

represent TPs of chlordecone. NMR analyses combined with chemical derivatization allowed to

402

structurally elucidate these TPs and the most significant TPs from families A and B (Figures S4).

403

Among them, the previously described “unknown nonpolar and polar products” reported by

404

Jablonski et al. in 199639 were assigned here according to thin-layer chromatography analysis to

405

TPs from families B and C, respectively (Figure S1). The present work thus significantly

406

expands the list of fully characterized chlordecone TPs, which hitherto only comprised

407

chlordecol, 8-monohydrochlordecone, and 2,8-dihydrochlordecone38, and also corrects previous

408

findings40-42.

409

In total, four of the five TP families (B, C, D and E) exhibit a rare indene aromatic bicycle

410

resulting from the opening of the chlordecone bishomocubane structure. Formation of the indene

411

ring correlated with the presence of vitamin B12 in chemical degradation experiments. It is

412

noteworthy that the only two isolated bacteria capable to transform chlordecone into such

413

indene-based TPs, i.e. Citrobacter sp. 86 and Citrobacter sp. 92, encode the full anaerobic

414

corrinoid biosynthetic pathway36. Indeed, corrinoids including vitamin B12 are known to act as

415

cofactors for reductive dehalogenases48-50. In their free form, they could also mediate reductive

416

dehalogenation under both biotic and abiotic conditions41, 51, 52. However, similarities between

417

the chemical and microbial ring-opening pathways could not be confirmed based on carbon

418

isotope fractionation37. This may be explained by the conditions used in chemical degradation

419

experiments, which are not relevant to those prevailing in microbial experiments.

420

Natural chlordecone degradation on Martinique Island


23

Page 25 of 33


421

Here, we assessed the presence of hydrochlordecones and polychloroindenes, as well as newly

422

identified chlordecone TPs, in several environmental compartments contaminated with

423

chlordecone sampled from Martinique Island. These included the Galion River basin (water, bed

424

sediments, and mangroves)29,

425

pumice stones (Table S6). Samples were analyzed using a different extraction protocol than the

426

recommended ISO 17025 standard31, 54, 55 to preserve their chemical composition. Specifically,

427

the initial drying step in soil analysis was omitted to limit volatilization of B, D, and E TPs.

428

Extracted samples were concentrated and analyzed by GC-MS and LC-HRMS in full-scan mode.

429

The only chlordecone derivatives previously detected in FWI soils were chlordecol and 8-

430

monohydrochlordecone, which were reported at much lower concentrations than chlordecone

431

(0.03-0.5 mg/kg, and 0.05-0.2 mg/kg, respectively)33, 44. These two compounds are also known

432

contaminants of commercial chlordecone. Thus, they have been the only chlordecone derivatives

433

included in targeted analyses until today. Although our dual GC-MS- and LC-HRMS-based

434

untargeted approach was intrinsically less sensitive than previous targeted methods, it enhanced

435

the robustness of TP assignment using isotopic patterns, and also allowed the detection of further

436

additional chlordecone TPs not present in our laboratory-based TP library.

437

We estimated the concentrations of the most abundant TPs (A1, chlordecol, B1, C1-C2, and C3-

438

C4) by external calibration with purified TP standards (Figure S11). We observed significant

439

variability in extraction efficiency from soils, depending on both TP and matrix, as shown for

440

chlordecone and metabolite B1 in andosol and nitisol soils (Figure S10). Thus, we provide only

441

uncorrected concentration ranges here.

442

Concentrations of chlordecone in the Galion River were in the range of previous studies (0.1-2

443

g/L)29. Of note, the concentration of TP B1 was in the same order of magnitude as that of

53

, andosol, nitisol, and ferralsol soils46,

54

, as well as ash and


24


Page 26 of 33

444

chlordecone itself (0.1-2 g/L) in both the Galion River and nearby mangroves (Figure 5 and

445

Table S6). Moreover, we detected TP B1 in bed sediments, in which chlordecone was not found.

446

Chlordecone concentrations in soils were approximately 10–fold lower than previously reported

447

typical values of dried samples (0.01-1 mg/kg compared to 0.8-5 mg/kg (dry weight46,

448

Chlordecone TPs were easily found in solid matrices, with B1 and chlordecol detected in all soil

449

samples, except in chlordecone-free nitisol 926. Concentration of TP B1 varied between 0.05 and

450

5 mg/kg. In contrast, chlordecol concentration was systematically around 0.05 mg/kg, in

451

agreement with previous studies44. We only observed significant levels of TP A1 in the two

452

andosol soils (0.05-1 mg/kg). TPs C3-C4, less chlorinated than C1-C2, were more frequently

453

detected, and at higher concentrations, in one nitisol soil (above 1 mg/kg). The low

454

concentrations of 8-monohydrochlordecone detected here (< 0.01 mg/kg) are also in agreement

455

with previous studies33. Worthy of note, we additionally detected many other TPs at low levels,

456

including

457

polychloroindenecarboxylates (E1-E2) (Figure 5 and Table S6). Our untargeted analytical

458

approach also led to the discovery of a monohydrochlordecol derivative and two dichloroindene

459

carboxylic acids (C7-C8) (Figure S4). These compounds had never been observed before in

460

laboratory biodegradation experiments (Figure S16 and Table S6). In contrast, we did not detect

461

methylated polychloroindenecarboxylates in any sample.

di-,

tri-hydrochlordecones,

tetrachloroindenes

(B2,

B3-B4),

and

54

).

ethyl


25

Page 27 of 33


462 463

Figure 5. Location of soil, water and sediment samples in Martinique and chlordecone and TP

464

concentrations.

465

In summary, this study demonstrates that four of the five known TP families, identified

466

previously and in this work through microbial and chemical laboratory experiments, are indeed

467

present in soils and water samples from Martinique. These four TP families include a total of 17

468

TPs not found in chlordecone commercial formulations (Figure S16). Here, pentachloroindene

469

B1 was the most prominent TP, with levels similar to those of chlordecone itself (Figure 5).

470

In addition, we also searched for bacteria affiliated to Citrobacter, shown previously to

471

transform chlordecone into TPs of A and B families36, in the studied soil samples, by Illumina


26


Page 28 of 33

472

sequencing of amplicons of the universal 16S rRNA gene (V4-V5 region) following DNA

473

extraction. Only a very low frequency (1.4 to 2.10-4) of Citrobacter-related sequences was found,

474

and this in only three of the 12 investigated samples (918, 919, and 920). Thus, the diversity of

475

bacteria associated with chlordecone degradation in Martinique soils has not yet been uncovered,

476

and likely extends beyond chlordecone-degrading organisms identified so far.

477

Along the same lines, we also set up soil/liquid microcosms under anoxic conditions to confirm

478

the widespread potential of native FWI soil microbiota to degrade residual chlordecone with

479

three representative FWI soils contaminated with chlordecone, i.e. one andosol (914), one nitisol

480

(918), and one ferralsol (919). Chlordecone degradation was observed in all experiments, with

481

concomitant formation of TPs (Figure S9), confirming intrinsic natural chlordecone degradation

482

in all investigated FWI soils, as well as production of several novel chlordecone TPs that had

483

hitherto remained undetected. The composition of indigenous bacterial communities will deserve

484

further investigation in the future.

485

Implication of natural chlordecone degradation

486

Overall, our results raise the question of the true extent of chlordecone pollution in the FWI.

487

Such contamination clearly extends beyond the parent chlordecone molecule to include several

488

previously undetected TPs. The structural diversity of TPs identified here illustrates the hitherto

489

unsuspected complexity of processes and pathways of chlordecone degradation in the

490

environment. Although chemical degradation experiments were not performed under

491

environmentally relevant conditions, they furnished a large panel of TPs, including those

492

detected in FWI, which were further purified and fully elucidated. Moreover, phylogenetic

493

analyses of bacterial communities suggest strong differences between FWI soil, sediment, and

494

river compartments and the previously isolated bacteria associated with chlordecone degradation.


27

Page 29 of 33


495

Thus, it cannot be excluded that yet other, still undetected, chlordecone TPs may also be formed

496

under field conditions. The results of the present prospective analytical campaign now warrant a

497

much more thorough study of the environmental relevance of these TPs and the associated biotic

498

and/or abiotic degradation processes of chlordecone. Indeed, the paradigm of absolute

499

chlordecone persistence, taken for granted for decades31, now clearly appears obsolete and calls

500

for setting new monitoring and risk priorities. Access to the new panel of chlordecone TPs

501

defined by the microbial and chemical laboratory approaches used in this work opens the door to

502

accurate environmental quantification protocols, toxicological studies, and biodegradation assays

503

of chlordecone TPs. Finally, the novel untargeted dual GC-MS- and LC-HRMS-based approach

504

used here may also be applied to other POPs of high concern, such as mirex, which shares the

505

same perchlorinated bishomocubane structure as chlordecone, to assess their global

506

biodegradability in natural environments.

507

ASSOCIATED CONTENT

508

Supporting Information.

509

The Supporting Information is available free of charge on the ACS Publications website at DOI:

510

Additional information on analytical, purification and chemical protocols, structural elucidation,

511

microbiological experiments, natural samples from Martinique Island, mass spectra, NMR

512

spectra (PDF)

513 514

AUTHOR INFORMATION

515

Corresponding Author


28


516

* E-mail: [email protected] or [email protected].

517

Author Contributions

518

1

M.C. and O.D.N. contributed equally

519

2

S.C., A.B. and D.M. also contributed equally

520

Notes

521

The authors declare no competing financial interests.

Page 30 of 33

522 523

ACKNOWLEDGMENTS

524

Support was provided by the INRA AIP Demichlord part of “Plan Chlordecone”, Commissariat à

525

l’Energie Atomique et aux Energies Alternatives (CEA), the Centre National de Recherche

526

Scientifique (CNRS) and the University Evry Val d’Essonne (UEVE). MC work was funded by

527

CEA and ODN work was supported by the "IDI 2017" project funded by the IDEX Paris-Saclay,

528

ANR-11-IDEX-0003-02. We also thank Magalie Lesueur-Jannoyer and Cécile Fischer for

529

helpful discussions; Olek Maciejak for assistance in NMR data collection; and Grégoire David,

530

Eddy Elisée, Stéphanie Fouteau, Caroline Menguy, Charles Mottes, Tiffany Prevost and Luc

531

Rangon for technical assistance. Institut de Chimie des Substances Naturelles and UEVE are

532

acknowledged for NMR and MS facilities, and The Région Ile de France and CNRS for their

533

contributions to acquiring NMR equipment.

534 535

REFERENCES


29

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536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581


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