Ronan Cariou

Paula Méndez-Fernandez

Sébastien Hutinet

Yann Guitton

Florence Caurant

Bruno Le Bizec

Jérôme Spitz

Walter Vetter∥

Gaud Dervilly

Non-targeted LC/ESI-HRMS detection of polyhalogenated compounds in marine mammals stranded on French Atlantic coasts

Keywords: Halogenated organic compounds, Non-targeted screening, Marine top predator, Environmental contaminant, Halogenated Natural Product, Heptachloro-1,2′-bipyrrole

Up to now, non-targeted analysis (NTA) of halogenated organic compounds in biota has mostly been performed using GC/MS based instruments. We intended to broaden the spectrum of physicochemical properties of amenable substances by taking advantage of liquid chromatography-high resolution mass spectrometry coupling fitted with electrospray ionisation source. Thus, a NTA strategy was applied to a set of twelve blubber samples belonging to five marine mammal sentinel species stranded on the French Atlantic coasts. It involved specific postacquisition data interpretation using open-source software HaloSeeker 1.0 for annotating chemical formulas. A total of 135 distinct unequivocal molecular formulas were assigned to 466 polyhalogenated ion clusters of interest. The most intense representative ions were identified as bioaccumulative heptachloro-1,2′-bipyrrole (Cl 7 -BP), α-hexabromocyclododecane and (1R,2S,4R,5R,1'E)-2-bromo-1-bromomethyl-1,4-dichloro-5-(2'-chloroethenyl)-5methylcyclohexane (MHC-1) in the μg/g lipids weight range. To the best of our knowledge, it is the first time that Cl 7 -BP, the most intense signal observed, is reported in biota. A dozen other compound families will require further in-depth work to gain structural information.

INTRODUCTION

The Stockholm Convention is a global treaty aiming at protecting human health and the environment from Persistent Organic Pollutants (POPs). [START_REF]Final Act of the Conference of Plenipotentiaries on the Stockholm Convention on Persistent Organic Pollutants; United Nations Environment Programme[END_REF] So far, about 30 anthropogenic polyhalogenated substances are listed in its annexes although (poly)halogenation is not a prerequisite for classification as a POP. Assumedly, many other substances might deserve more consideration. 2 Knowing what to investigate turns out to be a crucial question to guide future research and policy priorities, especially when considering potential (bio)transformation products.

For instance, the European Marine Strategy Framework Directive aims at implementing a precautionary approach for managing pressures on marine waters. 3 However, regarding contaminants, the Directive focuses on legacy pollutants. The future challenge will be the monitoring of main compounds of emerging concern. Before prioritising the compounds to focus on, based notably on their adverse effect, it would be ideal to have a holistic overview of the chemical exposome. [START_REF] Rappaport | Implications of the exposome for exposure science[END_REF] Due to their high trophic position, longevity and low capacity to biodegrade POPs, [START_REF] Borrell | Organochlorine concentrations declined during 1987-2002 in western Mediterranean bottlenose dolphins, a coastal top predator[END_REF][START_REF] Tanabe | Capacity and mode of PCB metabolism in small cetaceans[END_REF] marine mammals bio-accumulate and bio-magnify large amounts of these contaminants and serve as sentinels for assessing the quality of marine environments. [START_REF] Wells | Bottlenose dolphins as marine ecosystem sentinels: developing a health monitoring system[END_REF][START_REF] Bossart | Marine mammals as sentinel species for oceans and human health[END_REF] The occurrence of high levels of POPs has been associated with pathological uterus lesions, a disease complex including lesions on skin, claws, intestines, kidneys and adrenal glands, as well as immunosuppression. [START_REF] Bergman | Pathology of Baltic grey seal and ringed seal females with special reference to adrenocortical hyperplasia: is environmental pollution the cause of a widely distributed disease syndrome? Finn[END_REF][START_REF] Jepson | Relationships between polychlorinated biphenyls and health status in harbour porpoises (Phocoena phocoena) stranded in the United Kingdom[END_REF][START_REF] Kannan | Toxicity reference values for the toxic effects of polychlorinated biphenyls to aquatic mammals[END_REF] Therefore, identifying halogenated organic compounds (HOCs) of emerging concern, seeking degradation products of POPs and understanding their fate have become a major scientific activity in contaminants monitoring. [START_REF] Shaul | Nontargeted biomonitoring of halogenated organic compounds in two ecotypes of bottlenose dolphins (Tursiops truncatus) from the Southern California Bight[END_REF] In addition to anthropogenic pollutants, halogenated natural products (HNPs) are also of special concern. Over 5000 HNPs, mainly produced by marine sponges, algae and bacteria, have been described, [START_REF] Gribble | Occurrence of halogenated alkaloids[END_REF] some of which are persistent, bioaccumulative and/or bioactive. [START_REF] Vetter | Marine halogenated natural products of environmental relevance[END_REF] Several classes of environmental relevance have been repeatedly detected over the past 25 years. They contain mostly up to seven halogens, mostly brominated or Cl/Br-mixed. [START_REF] Vetter | Marine halogenated natural products of environmental relevance[END_REF] Targeted analysis remains state of the art for monitoring known contaminants of concern.

However, harmful but unknown compounds are inevitably missed. [START_REF] Shaul | Nontargeted biomonitoring of halogenated organic compounds in two ecotypes of bottlenose dolphins (Tursiops truncatus) from the Southern California Bight[END_REF] Arising from the advent of modern high-resolution mass spectrometry (HRMS) technologies, non-targeted analysis (NTA) workflows aim at broadening the scope of analysis by taking advantage of selective full scan mass spectra. While their advantages (e.g. accelerating the rate of contaminant discoveries) and weaknesses (e.g. reproducibility) are still controversially discussed, [START_REF] Hollender | Nontarget screening with high resolution mass spectrometry in the environment: ready to go?[END_REF][START_REF] Hites | Is nontargeted screening reproducible?[END_REF][START_REF] Samanipour | Letter to the Editor: Optimism for nontarget analysis in environmental chemistry[END_REF][START_REF] Ulrich | EPA's non-targeted analysis collaborative trial (ENTACT): genesis, design, and initial findings[END_REF] practices are rapidly evolving. Besides analytical chemist choices as regards sample preparation and data acquisition technique, the bottleneck shifted to post-acquisition data interpretation, requiring sophisticated cheminformatics tools to process huge datasets. Thus, human expertise remains essential to critically review and discuss the results.

Since most HOC of environmental relevance are non-polar, gas chromatography-based techniques (GC) initially appeared as the most suited strategy for tracking them. [START_REF] Vetter | Marine halogenated natural products of environmental relevance[END_REF] GC was either combined with electron capture and nitrogen-phosphorus detectors (ECD and NPD), electron capture negative ion mass spectrometry (ECNI-MS), electron ionization mass spectrometry (EI-MS), or more recently comprehensive two-dimensional GC coupled to time-of-flight mass spectrometry (GC×GC/EI-TOF/LRMS). [START_REF] Shaul | Nontargeted biomonitoring of halogenated organic compounds in two ecotypes of bottlenose dolphins (Tursiops truncatus) from the Southern California Bight[END_REF][START_REF] Vetter | Marine halogenated natural products of environmental relevance[END_REF][START_REF] Fujii | Tissue-specific bioaccumulation of long-chain perfluorinated carboxylic acids and halogenated methylbipyrroles in Dall's porpoises (Phocoenoides dalli) and harbor porpoises (Phocoena phocoena) stranded in northern Japan[END_REF] However, monitored HOCs excluded chemicals of intermediate polarity. Haraguchi et al. [START_REF] Haraguchi | Negative APCI-LC/MS/MS method for determination of natural persistent halogenated products in marine biota[END_REF] previously developed a targeted method dedicated to the heptachlorinated 1´-methyl-1,2′-bipyrrole (MBP) and a mixed hexahalogenated 1,1´-dimethyl-2,2´-bipyrrole (DBP) by liquid chromatography coupled to tandem mass spectrometry (QqQ) with atmospheric pressure chemical ionisation (LC/APCI-MS/MS), showing that atmospheric pressure ionisation coupled to LC was applicable to such compounds. More recently, Liu et al. [START_REF] Liu | Hundreds of unrecognized halogenated contaminants discovered in polar bear serum[END_REF] applied a non-targeted LC/ESI-HRMS method to investigate halogenated compounds in polar bear serum.

In order to better understand the fate of transformation products or to discover undescribed HOCs, whether of anthropogenic or natural in origin, we intended to further broadening the spectrum of physicochemical properties of amenable substances by taking advantage of liquid chromatography-mass spectrometry couplings (LC/MS) fitted with atmospheric pressure ionisation techniques.

In the present work, a robust NTA strategy involving LC/HRMS fitted with electrospray ionisation (ESI) was applied to characterise blubber samples of twelve individuals from five marine mammal species, including cetaceans and seals stranded on the French Atlantic coasts.

Because several biological (e.g. age and sex) and ecological factors (e.g. trophic level and prey type) affect the pollutant burden in marine mammals, [START_REF] Tanabe | Transplacental transfer of PCBs and chlorinated pesticides from the pregnant striped dolphin (Stenella coeruleoalba) to her fetus[END_REF][23][START_REF] Borrell | Evaluation of toxicity and sex-related variation of PCB levels in Mediterranean striped dolphins affected by an epizootic[END_REF][START_REF] Borrell | Differences in DDT and PCB residues between common and striped dolphins from the southwestern Mediterranean[END_REF] the species were selected with different feeding strategies (e.g. fish vs. cephalopods eaters) and habitats (e.g. inshore, offshore) meaning different environmental pollution exposures. Then, the objective was to characterize in these samples amenable non-conventional and known polyhalogenated organic compounds, using a recently made publicly available open-source software, HaloSeeker 1.0, for annotating chemical formulas and select signals worth further investigation. [START_REF] Léon | HaloSeeker 1.0, a user-friendly software to highlight halogenated chemicals in non-targeted high resolution mass spectrometry datasets[END_REF][START_REF] Chilczuk | Halogenation-guided chemical screening provides insight into tjipanazole biosynthesis by the cyanobacterium Fischerella ambigua[END_REF] The perspective is to enlarge the knowledge of the HOC exposome. EXPERIMENTAL A glossary of selected terms, abbreviations and chemicals is available in the Supporting Information. were provided by Cambridge Isotope Laboratories (Tewksbury, MA, USA). Pure (1R,2S,4R,5R,1'E)-2-bromo-1-bromomethyl-1,4-dichloro-5-(2'-chloroethenyl)-5methylcyclohexane (MHC-1) was previously isolated from red seaweed Plocamium cartilagineum. [START_REF] Vetter | Structure and origin of the natural halogenated monoterpene MHC-1 and its concentrations in marine mammals and fish[END_REF] The 2,3,3´,4,4´,5,5´-heptachloro-MBP (Q1 or Cl 7 -MBP) was synthesized according to Wu et al., [START_REF] Wu | Structure and synthesis of the natural heptachloro-1'-methyl-1,2'-bipyrrole (Q1)[END_REF] while the 3,3´,4,4´,5,5´-hexachloro-DBP (Cl 6 -DBP) and the 3,3´,4,4´tetrabromo-5,5´-dichloro-DBP (BC-10 or Br 4 Cl 2 -DBP) were synthesized according to Martin et al. [START_REF] Martin | Transition metals in organic synthesis, Part 97:1 silvercatalyzed synthesis of hexahalogenated 2,2′-bipyrroles[END_REF] A synthesis raw product (purity ~70%) of 2,3,3´,4,4´,5,5´-heptachloro-1,2′-bipyrrole (desmethyl-Q1, DQ1 or Cl 7 -BP), was produced by silylation of pyrrole, coupling with N-chlorosuccinimide, chlorination with POCl 3 /PCl 5 , removal of the protection group and initial chromatographic purification (Wu et al., in prep). Solvents and other chemicals used for sample preparation are described in the Supporting Information.

Chemicals

Samples. Blubber samples of twelve male (individual) marine mammals stranded on the Atlantic coast (English Channel or Bay of Biscay) between 2001 and 2018 were selected within the French sample bank of the national stranded marine mammals network (stored at -20 °C). Females were not considered in order to avoid any bias linked to the transfer of POPs to their offspring during gestation and especially lactation. Therefore, for this pilot study, two adult males from five species with very different feeding ecologies were selected, without any previous knowledge on the body burden of environmental contaminants: Phocoena phocoena (Pp, harbour porpoise), Balaenoptera physalus (Bp, fin whale), Tursiops truncatus (Tt, bottlenose dolphin), Phoca vitulina (Pv, harbour seal) and Physeter macrocephalus (Pm, sperm whale) (Table 1). Additionally, two harbour porpoise individuals (Pp_2 and Pp_3) exhibiting relatively high levels of legacy POPs were included to enrich the analytical fingerprints. These two samples belonged to another set of samples (harbour porpoise, n = 66) for which a set of POPs were previously quantified in blubber (unpublished results) according to ISO 17025 accredited methods. Harbour porpoise Pp_2 exhibited the highest level of dioxin-like polychlorinated biphenyls (dl-PCBs, OMS-TEQ2005 = 216 pg/g lipids weight, lw) and non-dioxin-like PCBs (∑ 6 ndl-PCBs = 199 µg/g lw) while harbour porpoise Pp_3 the highest level of polybromobiphenyl 153 (PBB-153, 83 ng/g lw). Details concerning stranding and animal sizes are provided in Table S1. 13 C 12 -γ-HBCDD as internal standard (IS). The extract was partitioned with 1.5 mL concentrated sulphuric acid added dropwise to remove lipids. Although it was previously found that removal of lipids by gel permeation chromatography (GPC) only, as opposed to acid treatment, resulted in the maximum number of detected compounds, [START_REF] Shaul | Nontargeted biomonitoring of halogenated organic compounds in two ecotypes of bottlenose dolphins (Tursiops truncatus) from the Southern California Bight[END_REF] we applied acid treatment in order (i) to minimise blank levels and (ii) to maintain the heaviest and most recalcitrant compounds. [START_REF] Vetter | Thorough study of persistent organic pollutants and halogenated natural products in sperm whale blubber through preparative sample cleanup followed by fractionation with countercurrent chromatography[END_REF] In turn, one must bear in mind that the observed signals correspond either to acid-insensitive or to degradation reaction products. After centrifugation (350 g, 10 min), the organic layer was separated and the procedure repeated three times. The limpid organic layer was neutralized twice with 2 mL water (up to pH = 3) and dried with anhydrous sodium sulphate. The extract was concentrated at 40 °C under a gentle stream of nitrogen and transferred to a vial containing 20 µL toluene with 10 ng 2 H 18 -β-HBCDD as recovery standard (RS). Finally, the extract was reconstituted in methanol/water 4:1 (v/v, 62.5 µL). Three procedural blanks were performed in parallel and, in addition, three vials containing IS and RS mixture were injected as well within the analytical sequence.

Data acquisition. Extracts (7 µL) were analysed in a single sequence with an UltiMate 3000 Post-acquisition data-treatment. Targeted peak integration of labelled IS and RS as well as MS/HRMS data interpretations were performed using Xcalibur software (Thermo Fisher Scientific). Non-targeted data mining on one-dimensional LC-HRMS datasets was performed using HaloSeeker 1.0, an open source software aiming to seek halogenated signatures in full scan HRMS fingerprints described by Léon et al. [START_REF] Léon | HaloSeeker 1.0, a user-friendly software to highlight halogenated chemicals in non-targeted high resolution mass spectrometry datasets[END_REF] The ergonomic web user interface in the R-programing environment avoids any interactions with the coding component while allowing interactions with the data, including peak detection, deconvolution, and comprehensive manual review for chemical formula annotation. Briefly, proprietary raw data were converted in an mzXML open format using the MSConvert 32 software (proteowizard version 3.0.9810). Then, the application proceeded to the peak picking step using xcms 3.2.0 package 33 (mzTol=3, snthresh=10, prefilter step=3, peakwidth=10-60, prefilter level=20,000, noise=0, mzdiff=0.001). Obtained features were paired according to precise mass differences between naturally occurring C, Cl and Br isotopes, according to a script adapted from Cariou et al. [START_REF] Cariou | Screening halogenated environmental contaminants in biota based on isotopic pattern and mass defect provided by high resolution mass spectrometry profiling[END_REF] , reconstituting halogenated isotopic patterns in so-called clusters (tolerances: t R =1 s, m/z=0.5 mDa). H/Cl-scale mass defect plots (MDplot) were drawn, using the data filters. Indeed, HaloSeeker 1.0 classifies features and clusters according to four filters. Among all features (F0 filter), those which are paired according to precise mass differences between naturally occurring C, Cl and Br isotopes belong to the F1 filter.

Considering the feature triplets including the base peak A and the adjacent A-2 and A+2 isotopologues, additional ion ratio rules restrict to F2 and F2+ filters, for halogenated and polyhalogenated clusters, respectively. [START_REF] Léon | HaloSeeker 1.0, a user-friendly software to highlight halogenated chemicals in non-targeted high resolution mass spectrometry datasets[END_REF] A graphical representation of the successive filters is available in the glossary of the Supporting Information. Cumulative intensity of features paired in a cluster (C ∑Int ) was used for comparisons. Finally, the clusters of interest were investigated using the interactive pop-up window offering a formula decomposition script adapted from the Rdisop package [START_REF] Böcker | A fast and simple algorithm for the money changing problem[END_REF] to consider either the peak of lowest m/z (likely the monoisotopic peak) or preferentially the most abundant isotopologue (likely the base peak). Chemical formulas were annotated by the (Figure S3). The singular presence of alkyl sulphates in sperm whale could be due to the very high level of wax (sterols and esters), the main lipid class in the blubber of this cetacean species [START_REF] Koopman | Phylogenetic, ecological, and ontogenetic factors influencing the biochemical structure of the blubber of odontocetes[END_REF] , and result from the incomplete oxidation by concentrated sulphuric acid.

IS and RS were not considered as representative of all amenable compound but rather as indicators of sample preparation and instrumental performances. Retention times (t R ) for IS and RS were stable along the LC-HRMS sequence. Mass deviations for both IS and RS were in the range 0.23-0.51 mDa (0.40-0.81 ppm) (Table S2), allowing for sub-ppm formula searches during the formula annotation step. IS recoveries in sample and blank extracts ranged from 50 to 93%, except for sperm whales (44-46%). These results were consistent with the fact that losses occurred at each partitioning, depending on the visual discernibility of the interface between n-hexane and concentrated sulphuric acid. Regarding sperm whales, lower recoveries are in agreement with the occurrence of the milky precipitates. Considering such losses and the ion suppression phenomenon, results were not considered quantitatively but qualitatively only. However, semiquantitative assumptions could be performed, assuming a recovery and a response factor similar to IS. All F2+ clusters were reviewed through the HaloSeeker interface and either discarded (n = 422, IS-or RS-related adducts or obviously non halogenated when considering extracted ion chromatograms -EICs, cluster slope, ion ratios) or annotated with an ion formula (n = 466, Table S4) according to all available information, including EICs, cluster's pattern and slope, pattern score and m/z deviation with theoretical suggestions. All this information allowed to reach level 4 of identification confidence (unequivocal molecular formula) according to the scale developed by Schymanski et al. [START_REF] Schymanski | Identifying small molecules via high resolution mass spectrometry: communicating confidence[END_REF] Among the 466 clusters, no cluster of interest other than labelled standards was detected in procedural blanks. In each blubber dataset, 4 to 94 clusters of interest were annotated with a polyhalogenated compound (Table 2). Compared to the total intensity of features (31-683×10 6 AU of cumulative C ∑Int ), they represented only 0.04 to 1.67% in sperm whale, harbour seal, fin whale and the two harbour porpoises randomly selected, compared to 4-8% in bottlenose dolphins and 12-24% in the two harbour porpoises contaminated with relatively high levels of legacy POPs.

Enumeration of clusters of interest

Although more samples are required to refine such levels and proportions, these differences support species-specific variabilities in the levels of polyhalogenated substances amenable by LC/ESI in blubber after acidic degradation. [START_REF] Wu | Structure and synthesis of the natural heptachloro-1'-methyl-1,2'-bipyrrole (Q1)[END_REF] They also support that high contamination levels with legacy POPs correlate with multi-exposures. [START_REF] Méndez-Fernandez | An assessment of contaminant concentrations in toothed whale species of the NW Iberian Peninsula: Part I. Persistent organic pollutants[END_REF][START_REF] Pinzone | POPs in free-ranging pilot whales, sperm whales and fin whales from the Mediterranean Sea: Influence of biological and ecological factors[END_REF] S5), for cumulative C ∑Int of 9.0 and 1.3×10 9 AU, respectively. Mass deviations for known compounds and annotated molecular formulas varied from 0 to 0.9 mDa but the observed distribution according to m/z suggested a lower confidence level for those formulas with mass deviation above 0.6 mDa (erroneous or suffering from interference) (Figure S6).

Remarkable cluster series.

After formula annotation, an exported data table was manually reviewed for grouping clusters mainly based on congeners, homologues, isomers, and adduct ions.

More than 300 out of the 466 clusters, corresponding to >90% of cumulative C ∑Int for each dataset, were grouped into 16 cluster series of decreasing intensity (Tables S5) gathering congeners and homologues. For each series, the most intense clusters among the datasets are displayed as MDplots in Figure 1, revealing patterns described hereafter.

+ C2H4O2 + H Cl + H NO3 # 1 # 2 # 3 # 4 # 5 # 6 # 7 # 8 # 9 # 1 0 # 1 1 # 1 2 # 1 3 # 1 4 # 1 5 # 1 6
[M -H] -pseudo-molecular ion of Cl 7 -BP was observed as losses of Cl and Cl 3 , cleavage of the Cl lw range in marine mammals worldwide. [START_REF] Shaul | Nontargeted biomonitoring of halogenated organic compounds in two ecotypes of bottlenose dolphins (Tursiops truncatus) from the Southern California Bight[END_REF][START_REF] Fujii | Tissue-specific bioaccumulation of long-chain perfluorinated carboxylic acids and halogenated methylbipyrroles in Dall's porpoises (Phocoenoides dalli) and harbor porpoises (Phocoena phocoena) stranded in northern Japan[END_REF][START_REF] Hauler | Discovery and widespread occurrence of polyhalogenated 1,1'-dimethyl-2,2'-bipyrroles (PDBPs) in marine biota[END_REF][START_REF] Alonso | Nontargeted screening of halogenated organic compounds in bottlenose dolphins (Tursiops truncatus) from Rio de Janeiro, Brazil[END_REF] They have been proven to biomagnify within marine food webs [START_REF] Tittlemier | Global distribution of halogenated Dimethyl bipyrroles in marine mammal blubber[END_REF][START_REF] Pangallo | Marine Natural Products, the halogenated 1′-methyl-1,2′bipyrroles, biomagnify in a Northwestern Atlantic food web[END_REF] and to be present in the human diet as well as in human milk. [START_REF] Tittlemier | Dietary exposure to a group of naturally produced organohalogens (halogenated dimethyl bipyrroles) via consumption of fish and seafood[END_REF][START_REF] Vetter | Determination of Q1, an unknown organochlorine contaminant, in human milk, Antarctic air, and further environmental samples[END_REF][START_REF] Vetter | Qualitative determination of bioaccumulative halogenated natural products in food and novel food[END_REF] Series #2 -α-HBCDD. The second most abundant compound detected in the investigated extracts was unambiguously identified as α-HBCDD using authentic standard (t R =15.40 min, C ∑Int ~0.9×10 9 AU in harbour porpoise Pp_3 extract, ΔmDa=0.34). HBCDD is a well-known POP widespread in the global environment [START_REF] Covaci | Hexabromocyclododecanes (HBCDs) in the environment and humans: a review[END_REF][START_REF] Law | Levels and trends of PBDEs and HBCDs in the global environment: Status at the end of 2012[END_REF][START_REF] Jondreville | Hens can ingest extruded polystyrene in rearing buildings and lay eggs contaminated with hexabromocyclododecane[END_REF] and one of the few POPs preferentially analysed by ESI. [START_REF] Omer | Enantiomer-specific accumulation and depuration of α-hexabromocyclododecane (α-HBCDD) in chicken (Gallus gallus) as a tool to identify contamination sources[END_REF] It was detected in all blubber extracts at concentrations in the 0.01 to 4 μg/g lw range. The technical product HBCDD consists of three major isomers with γ-HBCDD being the predominant one. [START_REF] Vetter | Determination of Q1, an unknown organochlorine contaminant, in human milk, Antarctic air, and further environmental samples[END_REF] In agreement with other studies, however, the α-isomer was the most abundant and the only HBCDD isomer detected in the samples. Cl 3 ) of the compound corresponded with the one of MHC-1, a HNP. [START_REF] Vetter | Determination and mass spectrometric investigation of a new mixed halogenated persistent component in fish and seal[END_REF] MHC-1, a monoterpene insensitive to concentrated sulphuric acid, [START_REF] Vetter | Structure and origin of the natural halogenated monoterpene MHC-1 and its concentrations in marine mammals and fish[END_REF] was previously reported in various marine environmental (including mammals) and food samples, as well as in human milk. [START_REF] Vetter | Structure and origin of the natural halogenated monoterpene MHC-1 and its concentrations in marine mammals and fish[END_REF][START_REF] Vetter | Non-polar halogenated natural products bioaccumulated in marine samples. II. Brominated and mixed halogenated compounds[END_REF][START_REF] Barón | Halogenated natural products in dolphins: brain-blubber distribution and comparison with halogenated flame retardants[END_REF] Subsequent LC/ESI-HRMS analysis of the harbour porpoise Pp_2 extract and MHC-1 standard solution resulted in a good match of retention times and MS/HRMS fragmentation patterns (Figure 2). Conversely, only

[C 2 H 3 O 2 ] -, [Br] -and [C 2 H 4 O 2 + Cl] -ions arose from the LC/ESI-MS/MS fragmentation of the [M + C 2 H 3 O 2 ] -adduct of MHC-1.
Of the five species studied, fin whale has the lowest trophic level. This species feeds almost exclusively on an invertebrate species, the euphausiid krill Meganyctiphanes norvegica, which lives in deep waters and hence may not be in proximity of the MHC-1 natural producer, the pelagic red seaweed Plocamium sp. [START_REF] Vetter | Structure and origin of the natural halogenated monoterpene MHC-1 and its concentrations in marine mammals and fish[END_REF] . Previous research indicated that the abundance of MHC-1 in the environment difficult to predict. [START_REF] Wu | Polyhalogenated compounds (halogenated natural products and POPs) in Sardine (Sardinops sagax) from the South Atlantic and Indian Oceans[END_REF] Here, MHC-1 concentrations were estimated up to 1.3 μg/g lw in harbour porpoise Pp_2. Interestingly, harbour porpoise Pp_2 and bottlenose dolphin Tt_1 extracts also featured a Br 3 Cl 2 homologue (t R =13.87 min) as acetate adduct at ~1% intensity relative to MHC-1. Both species showed a coastal habitat with a high trophic level among the species studied.

Other series. For series #4 to #16, carbon numbers ranged from 10 to 21 and DBE from 2 to 10.

All representative ions featured between 6 and 11 Cl, but no Br (Table 3). This could be due to the fact that Br-containing HNPs tend to be unstable in sulphuric acid. [START_REF] Vetter | Structure and origin of the natural halogenated monoterpene MHC-1 and its concentrations in marine mammals and fish[END_REF] Series #4 and #5 were remarkable because of their high number of carbons and masses which are at (or above) the limit of GC analysis. This may explain why no information could be found for these compounds in the consulted scientific literature. Series #4, #5 included mixed Cl/Br-containing homologues. This may indicate that they were also HNPs, [START_REF] Vetter | Marine halogenated natural products of environmental relevance[END_REF] while the other series would rather be of anthropogenic origin. Only three series originated from a single compound (#10, #12, #15), while the other indicated compound families of different complexity (e.g. #9 with 4 homologue groups and many isomers). Some series exhibited both pseudo-molecular and acetate adduct ions (#11, #13). MS 2 experiments were performed for selected representative ions (#4-9, #12), revealing that some were acetate adducts (#6-8, #12). Thus, attention must be paid to the fact that, beyond 2 oxygen atoms and considering the mobile phase, observed ions could correspond to acetate adducts, even if no co-eluting ion is observed. Eventually, at least 8 out of and less intense OH-PCBs and B9-1679 were identified at confidence level 1 (confirmed structure [START_REF] Schymanski | Identifying small molecules via high resolution mass spectrometry: communicating confidence[END_REF] ). On the other hand, compounds like polyhalogenated N-methyl-bipyrroles could not be detected by LC/ESI-HRMS. These results confirm that GC-and LC-based NTA methods are complementary, and that a range of polyhalogenated compounds will likely be overlooked when only one method is used. In this line, the water fraction used to neutralise the acidic extract may contain polar HOCs which could be investigated. Similarly, GPC would help investigating acidsensitive compounds.

The results of this study, including the very first reporting of Cl 7 -BP which turned out to be the most abundant polyhalogenated compound in the datasets, suggest that implementing this strategy would expand the knowledge of polyhalogenated compounds in the environment. Especially, for series #4 to #15, further in-depth work could help expressing and testing structural hypotheses based on interrogation of other databases as well as performing complimentary MS analyses on collected fractions, and at best comparing them with analytical standards. Also LC fractions could be collected and analysed by bioanalytical methods in order to assess the toxicology.

In terms of perspectives for the data mining, HaloSeeker could offer further automation and higher deconvolution by reconstituting mass spectrum (pairing co-eluted ions adducts/fragments) and/or by aligning clusters from one sample to another, opening the way to realistic investigation of long sequences with ionisation mode leading to complex spectra. A blank subtraction filter would also be valuable. Still, the manual review of deconvoluted data remains the best way to guarantee high quality output. Indeed, for example, although the parameters have been optimised

to minimise ghost peaks, it is likely that some will always occur and pass the filters. Connexions with internal and external databases would also help in annotating more rapidly signals from known compounds, including spiked standards.

.

  Hexabromocyclododecane (HBCDD) labelled isomers ( 13 C 12 -γ and 2 H 18 -β), Dechlorane 603 and five polychlorinated hydroxyl-biphenyls (OH-PCBs: 4-OH-pentaCB-107, 4-OH-hexaCB-130, 3-OH-hexaCB-137, 4-OH-hexaCB-146, 3-OH-hexaCB-153) were purchased from Wellington Laboratories (Guelph, Ontario, Canada). Three other OH-PCBs (3-OH-pentaCB-101, 4-OH-pentaCB-101, 5-OH-hexaCB-138) were obtained from AccuStandard (New Haven, CT, USA). Tetradecyl and hexadecyl sulphate sodium salts were obtained from Sigma-Aldrich (Saint-Louis, MO, USA). The 2-endo,3-exo,5-endo,6-exo,8,8,9,10,10-nonachlorobornane (Parlar 50 or B9-1679) and the 2,2,5,5,8,9,9,10,10-nonachlorobornane (Parlar 62 or B9-1025)

  UHPLC pumping system coupled to an Orbitrap Q-Exactive mass spectrometer equipped with a heated ESI source (Thermo Fisher Scientific, San Jose, CA, USA). The instrument was controlled with Chromeleon Xpress and Xcalibur software (Thermo Fisher Scientific). Chromatographic separation was performed on a C 18 -like analytical column (Hypersil Gold, 100 mm × 2.1 mm, 1.9 μm, Thermo Fisher Scientific) kept at 45 °C. A mobile phase consisting of acetonitrile/water 99:1 (v/v, A) and water (B), both containing 10 mM ammonium acetate, was used. The gradient started with 20% A (0 to 2 min), was then increased linearly to 60% (10 min), 100% (40 to 46 min) and returned to the initial conditions (48 to 52 min). The flow rate was set at 0.4 mL/min. Data were recorded in negative mode with ESI parameters as follows: sheath gas flow, 50 arbitrary unit (AU); auxiliary gas flow, 5 AU; capillary temperature, 350 °C; source temperature, 150 °C; spray voltage, 2.5 kV; s-lens radio frequency, 50 AU. One-dimensional HRMS data were acquired in full scan mode within the m/z range 120-1000 at a resolving power of 140,000 at m/z 200, using m/z 305.02307 ([CH 3 COO•(NaCH 3 CO 2 ) 3 ] -) as lock mass. The automatic gain control (AGC target) was set at 5×10 5 , and the maximum injection time (Max IT) was set at 250 ms. LC/ESI(-)-MS/HRMS fragmentation experiments were performed in the targeted-MS 2 mode according to the same chromatographic and ionisation parameters, with adjusted injection volume.Precursor ions were selected at ± 0.4 m/z and fragmented in the HCD cell at 10, 20, 40 and 60% of normalised collision energy (NCE). Mass spectra of fragments were recorded from m/z 50 on, with the following parameters: AGC target: 1×10 5 ; Max IT: 100 ms; resolution: 35,000 at m/z 200. GC/EI(+)-HRMS complimentary assays were performed on an Orbitrap Q-Exactive mass spectrometer coupled to a Trace 1310 GC equipped with a HT-5MS column (30 m × 0.25 mm, 0.25 µm, Restek, Bellefonte, PA, USA), controlled with Xcalibur software (Thermo Fisher Scientific). Solutions (2 µL toluene) were injected in the splitless mode at 300 °C. Helium was used as carrier gas at 1 mL/min. The oven temperature program ramped from 100 °C (2 min) to 325 °C (6 min) at 15 °C/min. Auxiliary and source temperatures were set at 325 and 300 °C, respectively, the electron energy at 70 eV. One-dimensional HRMS data were acquired in full scan mode (m/z 120-800) at a resolving power of 120,000 at m/z 200. AGC target was set at 5×10 5 and Max IT at 200 ms.

  user considering H, C, N, O, S, Cl and Br up to 50, 30, 10, 10, 10, 15 and 10 elements, respectively.The double bond equivalent (DBE) was deduced considering neutralisation with an additional proton. Ion ratio tolerance of the scoring tool was set at 20%. Theoretical centroid isotopic patterns were calculated according to Loos et al., 36 either through the package embedded in HaloSeeker or through enviPat Web 2.4, 37 using the ExactivePlus_R140000@200 resolution parameter.RESULTS AND DISCUSSIONTotal ion chromatograms and QA/QC. Aside from peaks of solvent front and of return to initial conditions, no signals were visible in the total ion chromatograms (TICs) of standards and procedural blanks injections (FigureS1). Unlike other studied species, sperm whale TICs exhibited intense signals. This is consistent with the lower purification degree revealed by the observed milky precipitate in the final vial requiring an additional centrifugation and separation step prior to injection. These interferences corresponded to[M -H] -ions of saturated alkylsulphates ([C n H 2n+1 O 4 S] -, 9 ≤ n ≤ 38) (Figures S2),as confirmed by standards of sodium n-tetradecyl-and n-hexadecylsulphate. Regardless of the chain length, MS 2 fragmentation featured the unique [HSO 4 ] -ion at m/z 96.9601 (± 2 ppm). EICs of this fragment ion showed the predominance of branched isomers which eluted prior to the corresponding n-alkyl sulphates

  and manual formula annotation. Using HaloSeeker 1.0 (see experimental), LC-HRMS datasets of marine mammals exhibited between 761 and 4,023 features above a threshold of 10 6 AU for C ∑Int . Procedural blanks and standards were quite variable, possibly due to the lack of initial sequence equilibration, lower ion suppression phenomenon and/or a threshold effect during the peak picking step. Corresponding MD-plots appeared consequently overloaded when considering all features (F0 filter) but pairing drastically decreased plots passing filter F1, as illustrated in FigureS4. Eventually, 47 to 98% of features and 74 to 99.9% of clusters were discarded between F0 (entire set of features) and F2+ (paired clusters passing the polyhalogenated ion ratio rules) filters, permitting the realistic manual review of 888 remaining polyhalogenated clusters we chose to focus on. Further details on enumeration of clusters of interest are available in the Supporting Information.

Figure 1 .

 1 Figure 1. H/Cl scale mass defect plot of most intense representative clusters among all marine

Figure 2 .

 2 Figure 2. Fragmentation patterns obtained for harbour porpoise Pp_2 base peak signals (Top) of

  However, none of these HNPs were detected in the samples analysed. A subsequent LC/ESI-HRMS analysis of standards of Cl 7 -MBP and Br 4 Cl 2 -DBP failed to give signals with the present method, obviously due to an inadequate ionisation technique. Therefore, the harbour porpoise Pp_2 extract was further analysed by GC/EI-HRMS on Cl 7 -BP, Cl 7 -MBP, Cl 6 -DBP and Br 4 Cl 2 -DBP, which were available as standards. Polyhalogenated Nmethyl-bipyrroles were unambiguously identified by GC/EI-HRMS, with Cl 7 -MBP (~1.7 μg/g lw) being about three order of magnitude more abundant than Cl 6 -DBP and Br 4 Cl 2 -DBP, but Cl 7 -BP (the predominant compound in LC/MS) could not be detected. Thus, a fraction centred about the LC retention time of Cl 7 -BP (±0.2 min) was collected using a switching valve placed between the LC analytical column and the ESI source. Re-analysis of this fraction by GC/EI-HRMS confirmed the presence of Cl 7 -BP in the harbour porpoise blubber. Matrix effect likely led to ion suppression by GC/MS in EI mode for the original harbour porpoise Pp_2 extract. It appears that Cl 7 -BP could be relatively sensitive to such a phenomenon. It might explain why Cl 7 -BP was not previously described in biota in GC-based studies, while ionization via cleavage of the N-H bond during ESI appeared credible. Assuming a response factor similar to IS, Cl 7 -BP concentration would reach 3.4 μg/g lw in harbour porpoise Pp_2. A pure analytical standard will be required prior to producing quantitative results and comparing with other polyhalogenated bipyrroles and HOCs.

  tR; shows HNO3 adduct; Cl9-, Cl8-, BrCl9-, BrCl8-and Br2Cl8-homologues; in-source fragments; analytical strategy combining LC/ESI(-)-HRMS and an open-source postacquisition data treatment software proved to be successful in highlighting the presence of abundant polyhalogenated substances in blubber of marine mammals. Precise mass information allowed determining 135 unequivocal molecular formulas for 466 ion clusters of interest, which were further grouped and reduced to 16 consistent series of compound classes. Literature searches allowed to hypothesise a few chemical structures. Interestingly, the compound spectrum detected via LC/ESI-HRMS in marine mammal blubber varied strongly from and was complimentary to the one described by NTA methods via GC/MS.[START_REF] Shaul | Nontargeted biomonitoring of halogenated organic compounds in two ecotypes of bottlenose dolphins (Tursiops truncatus) from the Southern California Bight[END_REF][START_REF] Vetter | Thorough study of persistent organic pollutants and halogenated natural products in sperm whale blubber through preparative sample cleanup followed by fractionation with countercurrent chromatography[END_REF][START_REF] Rosenfelder | Gas chromatography/electron ionization-mass spectrometry-selected ion monitoring screening method for a thorough investigation of polyhalogenated compounds in passive sampler extracts with quadrupole systems[END_REF][START_REF] Hoh | Nontargeted comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry method and software for inventorying persistent and bioaccumulative contaminants in marine environments[END_REF] Despite the knowledge of the molecular formulas, only part of the compounds could be tentatively annotated with known compounds, mostly known from GC analysis such as the B9-1679 nonachlorobornane (toxaphene), chlordane, OH-PCBs or Dechlorane 603 analogues. Eventually, the most intense Cl 7 -BP, α-HBCDD, MHC-1

Table 1 . Description of samples. BB: Bay of Biscay; EC: English Channel; Length in m. Previous analyses revealed relatively high levels of legacy POPs for harbour porpoise Pp_2 and Pp_3.

 1

	Species	Acronym	Collection year	Zone	Municipality	Length
	Phocoena phocoena	Pp_1	2012	BB	Tarnos	1.62
		Pp_2	2013	EC	Villiers-sur-Mer	1.54
		Pp_3	2015	EC	Dieppe	1.43

(500 mg) was suspended in 10 mL n-hexane and fortified with 20 µL toluene containing 20 ng

Table 2 .

 2 Enumeration , most being observed in several samples and some being isomers as well. The masses 286 detected by LC/ESI-MS ranged from m/z 203 to m/z 809. The compounds either primarily eluted from 8.4-21.3 min (less nonpolar range, 60-75% organic mobile phase) or from 29.6-35.0 min (most nonpolar compounds, 85-95% of organic mobile phase) (Figure

		FO		F2+ with annotated formula
	Sample	Features	Intensity	Features	Clusters	Total C ∑Int
	IS-RS_1	4,015	63,156	-	-	-
	IS-RS_2	671	9,933	-	-	-
	Blk_1	3,355	54,463	-	-	-

of features and clusters according to HaloSeeker filters. Feature and cluster intensities (C ∑Int ) as ×10 6 AU; C ∑Int threshold: 10 6 AU; t R range: 7-37 min; F0: all features; F2+: paired clusters complying polyhalogenated ion ratio rules.

  1,2'-bond with preferential charge retention on the Cl 4 -containing pyrrole moiety (C 4 Cl 4 N, monoisotopic peak m/z 201.8790), as well as [Cl 2 ] -ions. In addition, mixed halogenated homologues of Cl 7 -BP (Br 1 Cl 6 -, Br 2 Cl 5 -BPs) were also detected at slightly lower t R , with 9-14% and 1.6-2.0% LC/ESI-MS abundance relative to Cl 7 -BP, respectively. Several unresolved isomers were visible on EICs. This series accounted for more than 40% of cumulative C ∑Int in 3 out of 4 harbour porpoises.

  The third most abundant cluster observed in the study (t R =13.51 min, C ∑Int ~0.45×10 9 ) was detected in the harbour porpoise Pp_2 extract. When neutralised with a proton, the base peak at m/z 458.8542 corresponded to the formula [C 12 H 16 Br 2 Cl 3 O 2 ] -(ΔmDa=0.16, score=96%, DBE=2). This mixed pentahalogenated compound was also detected in all other extracts except those from fin whales. The LC/ESI-MS spectrum also featured a [C 10 H 12 Br 2 Cl 3 ] -ion (~4% relative intensity), suggesting a pair of acetate adduct and pseudo-molecular ions. Therefore, the deduced molecular formula (C 10 H 13 Br 2

	48
	Series #3 -MHC-1.

Table 3 .

 3 13 series contained 1 or 3 oxygen atoms according to LC/ESI-MS. Members of series #5 eluted between 29.6 and 35.0 min and exhibited pseudo-molecular and nitrate ions at similar relative intensities, with DBE=10. Slight in-source fragmentation was observed for the representative [C 21 H 17 Cl 10 ] -ion. MS 2 experiments confirmed the nitrate adduct and showed successive HCl losses down to [C 21 H 8 Cl 1 ] -. Successive HCl losses were observed for other series (#4, #10).Database searches in the CompTox Chemistry Dashboard 57 and SciFinder returned a few hits, Chlordene chlorhydrin has been reported as a biotransformation product of nonachlor and chlordane (see series #12) in terrestrial mammals and fish.[START_REF] Tashiro | Metabolism of Trans-Nonachlor and related Chlordane components in rat and man[END_REF][START_REF] Feroz | Metabolism, tissue distribution, and elimination of cis-[ 14 C]Chlordane in the tropical freshwater fish Cichlasoma sp[END_REF] Concerning series #7 and #8, Liu et al.[START_REF] Liu | Novel Dechlorane analogues and possible sources in peregrine falcon eggs and shark livers from the Western North Atlantic regions[END_REF] suggested the presence of a carbonylic derivative and a monohydro analogue of Dechlorane 603, using GC-MS instruments, in peregrine falcon eggs and shortfin mako shark livers from the Western North Atlantic regions. Within series #8, a homologue compound observed at 21.18 min (acetate adduct ion) exhibited the same ion formula as an acetate adduct ion of Dechlorane 603. Cl 9 O) corresponds to a hydroxylated toxaphene metabolite. This substance class, polychlorinated hydroxybornanes, was recently detected in fish.5861 Interestingly as well, ten series represented compounds with an even carbon number while six (#4, #5, #7, #8, #11, #13) had odd carbon numbers. Odd carbon numbers are rather unusual and uncommon for anthropogenic POPs. Summary of cluster series characteristics, including their most intense representative ions. t R : retention time; DBE: double bond equivalent when considering the neutral mass with an additional proton.

	allowing to increase confidence level up to 3 (tentative candidate) 37 for series #6 (chlordene
	chlorhydrin), #7 and #8 (carbonylic derivative and monohydro analogue of Dechlorane 603,
	respectively), #9 (polychlorinated hydroxyl-biphenyls (OH-PCBs) or diphenyl ethers, and a
	tetrabromo hydroxyl-diphenylether), #12 (chlordane) and #16 (nonachlorobornane). For series #6,
	Further experiments (full scan and MS 2 ) showed that Dechlorane 603 standard was amenable by
	LC/ESI-MS as acetate adduct ions. However, retention times did not match (5 min shift). Still,
	both compounds remain isomers. Eight authentic standards of OH-PCBs were injected and
	provided matches with 2 isomers (3-OH-pentaCB-101 and 3-OH-hexaCB-153), showing that
	series #9 corresponded to chlorinated hydroxyl-biphenyls (OH-tetra-to heptaPCBs). For series
	#16, nonachlorobornane was confirmed using authentic standard (level 1) in the bottlenose dolphin
	Tt_1 extract as B9-1679 (nonachlorobornane isomer), a component of the legacy POP toxaphene,
	by both LC/ESI(-)-HRMS and GC/EI(+)-HRMS. The presence of B9-1025 was also confirmed by
	GC/EI(+)-HRMS. Series #15 (C 10 H 9

ACKNOWLEDGMENT

The authors want to express their acknowledgments to all the members of the French national stranding network for their assistance in the field and data collection processes. Among reviewers, we particularly acknowledge reviewer #2 for the SciFinder search.

Funding Sources

This research was funded by the French General Directorate for Food as well as the European Union's Horizon 2020 research and innovation programme HBM4EU under Grant Agreement No. 733032.

Gachet, Nantes, F-44307, France. e-mail: laberca@oniris-nantes.fr (R. Cariou).

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.