Dr Saidbakhrom Saidjalolov
email: saidbakhrom.saidjalolov@etu.parisdescartes.fr

Dr Zainab Edoo
email: zainab.edoo@crc.jussieu.fr

Dr Matthieu Fonvielle
email: matthieu.fonvielle@crc.jussieu.fr

Dr Louis Mayer
email: mayer.louis@outlook.fr

Prof Laura Iannazzo
email: laura.iannazzo@parisdescartes.fr

Michel Arthur
email: michel.arthur@crc.jussieu.fr

Dr Mélanie Etheve-Quelquejeu
email: melanie.etheve-quelquejeu@parisdescartes.fr

Dr Emmanuelle Braud
email: emmanuelle.braud@parisdescartes.fr

Synthesis of carbapenems containing peptidoglycanmimetics and inhibition of the cross-linking activity

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The biosynthesis of peptidoglycan (PG), the major constituent of the bacterial cell wall, has been intensively studied to identify targets for drug development. A number of inhibitors of the constitutive enzymes have been described, [START_REF] Silver | [END_REF]2] the most successful as antibiotics being the βlactams. These compounds inhibit the enzymes that catalyze the final transpeptidation step in which adjacent glycan chains made of alternating N-acetylglucosamine (GlcNAc) and Nacetylmuramic acid (MurNAc) are linked to each other by formation of an amide bond between stem peptides carried by MurNAc residues (Scheme 1A). D,D-transpeptidases, commonly referred to as penicillin-binding proteins (PBPs), catalyze the formation of 4→3 cross-links connecting the fourth position of an acyl donor stem peptide to the side chain amino group of a di-amino acid located at the third position of an acyl acceptor stem peptide. Unusual 3→3 crosslinks formed by unrelated L,D-transpeptidases [3] (Ldts) (Scheme 1B) are predominant in mycobacteria [4] and in β-lactam-resistant mutants of Enterococcus faecium [5] and Escherichia coli [6] selected in vitro.

Scheme 1. Transpeptidation reactions with the PG subunit of E. faecium, catalyzed by A) PBPs and B) Ldts. C) Examples of drugs belonging to the three main classes of β-lactams. D) Formation of the covalent adduct (acylenzyme) resulting from nucleophilic attack of a carbapenem by the active-site serine of PBPs or the active-site cysteine of Ldts.

In the β-lactam family, members of the carbapenem class (imipenem, meropenem, ertapenem, and doripenem) have been developed and optimized for the treatment of infections due to Gramnegative bacteria producing extended-spectrum β-lactamases active on third-generation cephalosporins (Scheme 1C). [7] Optimization relied on introduction of various substituents at the C2 position and of a methyl at position C1. The latter modification increases the stability of the carbapenems with respect to hydrolysis by a host enzyme, albeit at the expense of a reduced reactivity of the drugs. [8] Carbapenems have been shown to be active against Mycobacterium tuberculosis in vitro [9,10] and in vivo [11] and are part of the recommended treatment of pulmonary infections due to Mycobacterium abscessus in cystic fibrosis patients. [12,13] The antibacterial activity of carbapenems against M. tuberculosis and M. abscessus involves inactivation of multiple targets including PBPs and Ldts. [14][15][16] The drugs irreversibly inactivate these enzymes by formation of a long-lasting covalent adduct resulting from nucleophilic attack of the βlactam carbonyl by the active-site serine of PBPs or by the active-site cysteine of Ldts (Scheme 1D). [17][18][19] β-Lactams are structure analogues of the PG precursors and act as suicide substrates of the transpeptidases. This conclusion was initially based on the pioneering work of Tipper and Strominger reported in 1965, who noticed that one of the possible conformations of the terminal D-Ala 4 -D-Ala 5 dipeptide of stem pentapeptide is similar to that of penicillin (Figure 1A). [20] By analogy, we present a carbapenem with a conformation that simulates the D-Ala 4 -D-Ala 5 (Figure 1A) or L-Lys 3 -D-Ala 4 (Figure 1B) extremity of the pentapeptide or tetrapeptide stem peptides, which are used as acyl donor by transpeptidases of the D,D and L,D specificities, respectively.

Several groups have investigated the possibility of modifying the structure of the substituents linked to the bicyclic core of β-lactams to improve drug binding and antibacterial activity. This was investigated not only with D,D-transpeptidases but also with other members of the "D,Dpeptidase" super family that share a common fold and catalytic mechanism but perform distinct functions in PG metabolism (Figure 2). This diversity is relevant to the current study since no general conclusion could be drawn for the various representatives of the D,D-peptidases subclasses. The premise of these investigations was that grafting groups resembling to PG precursors onto D-Ala-D-Ala and β-lactams should lead to parallel increases in the catalyzed acylation reaction rates with PG precursor analogues and β-lactams. The substituents of these peptidomimetics were designed to mimic the side or main chain of the PG precursors (in green and in blue in Figure 1C). Of note, the structure of the main chain is conserved whereas that of the side chain is variable in different bacterial species. Parallel increases in the acylation rates with peptidomimetic substrates and -lactams were observed for the R61 D,D-peptidase isolated form a bacterial strain belonging to Streptomyces genus (L,L-DAP-Gly side chain). R61 is a class C low-molecular weight D,D-peptidase of uncertain function with respect to PG metabolism. In vitro, purified R61 catalyzes formation of 4→3 cross-links (D,D-transpeptidase activity) and hydrolysis of D-Ala 5 (D,D-carboxypeptidase activity) by using as substrates synthetic peptides containing the terminal acyl-D-Ala-D-Ala moiety of PG precursors (Figure 1C). [22] The known structure of the PG of Streptomyces spp. implies that R61 functions in vivo with branched PG precursors composed of a linear stem peptide (L-Ala--D-Glx-L,L-DAP-D-Ala-D-Ala) and a side chain composed of a single Gly residue linked to the ε amino group of L,L-DAP (Figure 1C). [23] Pratt et al. explored the impact of grafting the L,L-DAP(Gly) dipeptide onto D-Ala-D-Ala and representative β-lactams of the penam (penicillin) and cephem classes (Figure 1C). Introduction of the peptidoglycan-mimetic side chain specific of the Streptomyces spp. led to dramatic parallel increases in the activity of the R61 D,D-peptidase with PG precursor analogues and in the reactivity of the enzyme with β-lactams. [21] Figure 1. A) Structural similarity between β-lactams (penams and carbapenems) and the pentapeptide donor stem of PBPs. B) Structural similarity between carbapenems and the tetrapeptide donor stem of Ldts. C) Structure of the peptidoglycan subunit used by the R61 D,D-peptidase in Streptomyces spp. and peptidomimetic penams. [21] The third position of the stem pentapeptide is occupied by an L,L-diaminopimelyl (DAP) residue substituted by a Gly residue. D) Structure of peptidomimetic carbapenems synthesized in this study. On the left: the substituents of the carbapenem core mimic the main chain of the PG subunit of E. faecium (shown in blue). On the right: the substituents of the carbapenem core mimic the side chain of the PG subunit of E. faecium (shown in green). R1 = -S(CH2)2Ph; R2 = H, L-Ala or L-Ala-D-Lac; R3 = H or CONH2; R4 = H, Me or NH2; R5 = H, Ac; n = 0-4. Inset: Structural analogy between carbapenem 28b and the L-Lys-D-iAsn moiety of peptidoglycan precursors. The inactivation rate constant of the R61 D,D-peptidase by the penicillin peptidomimetic containing the L,L-DAP(Gly) motif (1.5 x 10 7 s -1 .M -1 ) was three orders of magnitude higher than that of penicillin (1.4 x 10 4 s -1 .M -1 ), approached the diffusion limit, and was the largest rate constant yet reported for inactivation of a D,D-peptidase. The fact that the L,L-DAP(Gly) substituent of the L,L-DAP(Gly)-penicillin actually acted as a peptidomimetic was confirmed by the crystal structures of R61 in complex with L,L-DAP(Gly)-penicillin and L,L-DAP(Gly)-D-Ala-D-Ala. [24,25] These structures showed that the L,L-DAP(Gly) moieties of these molecules occupy nearly identical positions in the R61 catalytic cavity and form the same interactions with enzyme residues. These observations suggest that the "perfect" penicillin with respect to inactivation of the R61 D,D-peptidase was obtained by the peptidomimetic approach. [21] Similar results, although quantitatively less impressive, were obtained with other peptidomimetic specific of the various PG chemotypes but this only concerned class C D,D-peptidases [26][27][28][29] and the side chain of peptidoglycan precursors. [26,30,31] The group of Mobashery explored the bi-substrate approach by synthesizing a complex molecule consisting of a cephalosporin core substituted by mimics of the donor and acceptor stems of the transpeptidation reaction. Crystallization of the R61 D,D-peptidase acylated by this cephalosporin resulted in a model for the binding of the donor and acceptor substrates of the transpeptidation reaction. [32,33] The same approach adapted to the structure of the E. coli peptidoglycan structure led to a cephalosporin that behaved as a time-dependent and irreversible inhibitor of E. coli PBP1b, a Class A D,D-transpeptidase (first-order inactivation rate constant kinact of 0.0072 ± 0.0007 min -1 and inhibitor constant Ki of 2.5 ± 0.8 mM). [33] The peptidoglycan mimetic approach has only been applied to D,D-transpeptidases of the PBP family and to β-lactams of the penam (penicillin) and cephalosporin classes. This prompted us to design and develop the synthesis of carbapenem-based peptidoglycan mimetics and to evaluate their efficacy in the inactivation of the prototypic L,D-transpeptidase from E. faecium. [3,18,19] In the literature, the two main synthetic strategies providing access to functionalized carbapenems are either based on (i) the formation of the 4,5-fused nuclei from acyclic precursors (i.e. the carbapenem core including the ß-lactam ring) [34] or (ii) a direct addition of functionalities at the C2 and C8 positions on the preformed skeleton. [35] In this study, we took advantage of the nucleophilicity of the hydroxyl group at position C8 to connect various PG fragments via the formation of an ester bond. The resulting carbapenem-based peptidoglycan mimetics were divided into two categories containing portions of either the main chain or the side chain of the E. faecium PG subunits (Figure 1D, in blue and green, respectively). A functionalized carbapenem with a phenethylthio group at the C2 position was chosen as the model building block for modifications at C8 since previous work indicated that the latter led to effective inactivation of Ldts. [36]

Results and Discussion

Synthesis of carbapenem-based peptidoglycan mimetics (peptido-carbapenems).

The functionalized carbapenems were synthesized according to the retrosynthesis outlined in Scheme 2 starting from the commercially available -methyl-vinyl-phosphate 1. The phenethyl side chain was introduced at the C2 position by nucleophilic substitution of the phenylphosphate. [36] The protected amino acids or peptides mimicking various moieties of the stem peptide of PG precursors were incorporated via esterification of the C8 hydroxyl group. In this strategy, the p-nitrobenzyl (PNB) carbamate group was chosen as the main protecting group for the synthesis of peptidoglycan mimetics, enabling a single final deprotection step. Of note, the initial syntheses of the former derivatives conducted with benzyl and benzyloxycarbonyl protecting groups were unsuccessful since these groups could not be efficiently removed during the final deprotection step. Scheme 2. Retrosynthesis of peptido-carbapenems from 1.

The general procedure for N-CO2PNB protection were found to be highly efficient and versatile providing the protected amino acids and analogues 3 to 10 in 63% to 99% yield (Scheme 3A). D-iso-glutamine derivative 13 was prepared in a three-step procedure (Scheme 3B). The carboxylic acid of D-glutamic acid was selectively protected using trimethylsilyl chloride and methanol [37] followed by the introduction of the PNB group onto the amine affording amino acid 11 in 69% yield. The carboxylic acid was then activated with ethyl chloroformate and the reaction with aqueous ammonia provided compound 12 in 71% yield. In the last step, deprotection of the carboxylic acid was performed with LiOH affording the D-iso-glutamine derivative 13 in 87% yield. Peptidoglycan mimetic 16 was obtained by using as the starting material 4-aminobutyric acid, which was first converted into the corresponding methyl ester 14. [38] Coupling of 14 with alanine 8 assisted by K-oxyma® and EDC as coupling reagents afforded 15 in 86% yield. Deprotection of the methyl ester was performed with LiOH leading to the corresponding acid 16 in 63% yield (Scheme 4A). The synthesis of dipeptide derivative 20 started with the formation of the amino benzyl ester 17 as previously described. [39] Coupling with Boc-L-Ala-OH was achieved using TBTU as the coupling agent. Compound 18 was treated with TFA to remove the Boc group and reacted with (2R)-2-methoxypropanoic acid affording compound 19 in 71% yield. The final deprotection step was achieved by hydrogenolysis in methanol providing acid 20 in 76% yield (calculated by NMR) together with the corresponding methyl ester (Scheme 4B).

Scheme 4. Synthesis of peptidomimetics 16 (A), 20 (B) and 26 (C).

To obtain peptide 26 (Scheme 4C), the -benzylated D-glutamic acid 21 was protected with a Boc group affording 22 in 71% yield. Subsequent amidation afforded 23 in 96% yield. The N-Boc group of 23 was cleaved and coupling with Boc-L-Ala-OH afforded 24 in 70% yield.

Compound 24 was treated with TFA and coupling with (2R)-2-methoxypropanoic acid afforded to peptide 26 in almost quantitative yield.

The synthesis of peptido-carbapenems 28a-n was achieved in two steps (Table 1). Esterification of carbapenem 2 was first conducted with the PG analogues using a published procedure [40] affording carbapenems 27a-n in 35 to 99% yield. Finally, the peptido-carbapenems 28a-n were obtained by hydrogenolysis of 27a-n after HPLC purification. Using this approach, a full mimetic of the main chain was obtained (28n). In contrast, the best mimic of the side chain was 28b (inset in Figure 2D) since incorporation of the D-iAsn extremity of peptidoglycan precursor in the carbapenem could not be obtained due to cyclisation of D-iAsn during the synthesis of the L-Lys-D-iAsn dipeptide.

Efficacy of acylation of the Ldtfm L,D-transpeptidase by the peptidomimetic

carbapenems. Inactivation of L,D-transpeptidases was previously shown to be a two-step reaction [8] starting with nucleophilic attack of the β-lactam carbonyl by the sulfur of the catalytic cysteine (Scheme 5). This first step was proposed to lead to reversible formation of an amine anion. [41] The second step is irreversible, except for the cephalosporin nitrocefin, due to rupture of the β-lactam C-N bond followed by protonation of the nitrogen in the case of carbapenems. [42,43] Mass spectrometry analyses showed that all carbapenems synthesized in this study formed the expected acylenzymes without any secondary modification of the compound following the acylation step, as is the case for other classes of β-lactams (Table 2). [10,17] These results also indicated that the ester link at the C8 position was not hydrolyzed in the Ldtfm active site. This was further confirmed by incubating peptido-carbapenem 28g in the presence or absence of Ldtfm for 0, 90, and 240 minutes. Loss of 114,2 Da expected for the hydrolysis of the ester at C8 was observed in neither cases.

The efficacy of Ldtfm inactivation by peptidomimetic carbapenems was evaluated by determining the k1 and k2 kinetic constants for the formation of the amine anion and the acylenzyme (Scheme 5), respectively. [8,17,41,44] These kinetic constants were determined by stopped-flow fluorimetry as the intrinsic fluorescence of the Ldtfm tryptophan residues varies for the free enzyme, the amine anion, and the acylenzyme. [41] Fluorescence kinetics were determined with a minimum of three concentrations for each carbapenem. The rate constants k1, k-1 and k2 were determined by fitting differential equations to these kinetics using the DynaFit software, [45] as described in section 5 of the Supplementary Information. Peptidocarbapenems 28a to 28n harbored a phenethylthio substituent at position C2. In comparison to the reference carbapenem, meropenem, the phenethyl side chain improved both the k1 and k2 Scheme 5. Two-step acylation of L,D-transpeptidase Ldtfm by carbapenems. The evidence for the formation of amine anion in the first step of the acylation reaction is supported by previous studies. [8,43,44] Table 2. Mass of adducts formed between Ldtfm and carbapenems.

kinetic parameters (compare meropenem and compound 2' in Table 3). In comparison to 2', introduction of peptidomimetics at the C8 position had minor negative impacts on the k2 kinetic constant for all compounds (28a to 28n). Thus, the peptidomimetics reduced the efficacy of the chemical step of the reaction involving the rupture of the β-lactam ring and protonation of its nitrogen atom. The peptidomimetics had minor positive and negative impacts on kinetic constant k1, which is the critical constant determining the efficacy of Ldtfm inactivation. [41] These results show that substitutions at the C8 position marginally modulate the efficacy of inactivation of Ldtfm, as previously found for high-molecular weight PBPs.

Antibacterial activity. Peptido-carbapenems were tested by the microdilution technique in 96well plates against E. faecium strain M5 in the presence of ampicillin (32 µg/ml), as previously described. [START_REF] Peters | Proc. Natl. Acad. Sci. U. S. A[END_REF] Under such conditions, Ldtfm is the only functional transpeptidase due to inhibition of PBPs by ampicillin. [START_REF] Mainardi | [END_REF] The minimal inhibitory concentrations (MICs) of peptidocarbapenems 28a to 28n were equal to or higher than that of meropenem indicating that the peptidomimetic did not improve antibacterial activity.

Conclusion

We show that the C8 position of carbapenems can be efficiently functionalized by esterification to introduce chemical diversity in carbapenems. We added to this position mimics of the side chain (L-Lys-D-iAsn) and main chain (D-Lac-L-Ala-D-iGln-L-Lys) of peptidoglycan precursors from E. faecium. Biological evaluation of the resulting series of 14 peptidocarbapenems indicated that increasing similarity between the drug and the stem peptide of PG precursors does not improve the efficacy of inactivation of the L,D-transpeptidase produced by this species. Similar negative results were previously reported for other species-specific βlactam peptidomimetics designed to inhibit the transpeptidase activity of HMW class A and B PBPs (see introduction section). As proposed by Josephine et al., [27] this could be accounted for by the mode of substrate recognition by class A and B D,D-transpeptidases that may involve regions of the enzyme other than the transpeptidase active site and the glycan chains rather than the stem peptides. Such interactions were identified by solid-state NMR analysis of an L,Dtranspeptidase from Bacillus subtilis. [48] The carbapenems reported in this study were more effective than the reference drug of this class, meropenem, due to the positive impact of the phenethylthio substituent at position C2 combined to the mostly neutral impact of the substituents at position C8. Thus, the latter position can be modified to modulate the pharmacokinetic or other properties of carbapenems without any deleterious effect on the efficacy of L,D-transpeptidase inactivation or antibacterial activity.

This strategy could be used to optimize carbapenems for the treatment of lung infections due to Mycobacterium tuberculosis and Mycobacterium abscessus since L,D-transpeptidases are major contributors to peptidoglycan polymerization in these bacteria. [4,10,11,49,50] Table 3. Efficacy of acylation of the Ldtfm L,D-transpeptidase by meropenem and peptidocarbapenems and antibacterial activity against E. faecium M512.

[a] The methods used to determine kinetics parameters k1 and k2 are described in Section 4 of the Supplementary Information.

[b] Median from three independent experiments. [c] Synthesis of carbapenem 2' was previously described. [36] Cpd R1 R2 k1 (µM -1 min -1 ) [a] k2 (min -1 ) [a] MIC (µg ml -1 ) [

Figure 2 .

 2 Figure 2. Classes of

  Active-site Ser amidotransferases superfamily defined by a conserved fold and conserved SxxK, SDN, and KTG motifs

Scheme 3 .

 3 Scheme 3. A) Synthesis of N-CO2PNB carbamates protected compounds 3-10. B) Synthesis of compound 13.

Table 1 .

 1 Synthesis of peptido-carbapenems 28a-n.

	[a] Isolated yield. [b] Commercially available. [c] NMR calculated yield. [d] Isolated yield after HPLC
	purification.

  b]

	Meropenem	H	0.0819 ± 0.0004	1.59 ± 0.01	1
	2' [c]	H	0.292 ± 0.004	14.8 ± 0.18	2
	Side chain Mimetics
	28a		0.248 ± 0.004	5.10 ± 0.05	4
	28b		0.347 ± 0.010	4.68 ± 0.08	16
	28c		0.572 ± 0.007	7.40 ± 0.06	2
	28d		0.343 ± 0.009	3.92 ± 0.06	16
	28e		0.268 ± 0.003	7.49 ± 0.07	4
	28f		0.109 ± 0.001	3.16 ± 0.04	8
	28g		0.138 ± 0.002	3.43 ± 0.03	8
	28h		0.379 ± 0.005	5.20 ± 0.05	1
	28i		0.278 ± 0.005	3.80 ± 0.04	4
	28j		0.168 ± 0.002	2.70 ± 0.02	8
	Main chain Mimetics
	28k		0.239 ± 0.010	4.60 ± 0.11	4
	28l		0.192 ± 0.001	4.79 ± 0.03	4
	28m		0.355 ± 0.006	7.88 ± 0.11	8
	28n		0.121 ± 0.002	9.07 ± 0.15	> 16

Acknowledgements

The authors thank the "Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation" for S. S. PhD grant.

Experimental Section

General procedure for the synthesis of peptido-carbapenems 27a-n (Step 1). Carbapenem 2 (1 equiv.) in presence of peptidoglycan mimetic (4 equiv. up to 8 equiv.) was treated with DMAP (0.9 equiv.) and then with small portions of EDC (8 equiv. up to 16 equiv.) at -20 ˚C. The reaction mixture was stirred for 1 -24 hours while maintaining a temperature between -30 to -15 ˚C. After the completion of the reaction, EtOAc was added to dissolve the crude. The organic layer was washed with a sat. solution of NaHCO3, brine, dried over MgSO4, filtered and concentrated under reduced pressure. The crude mixture was purified by silica gel chromatography (cyclohexane:EtOAc or DCM:MeOH as eluent systems) to afford the desired peptido-carbapenem derivatives.

General procedure for the synthesis of peptido-carbapenems 28a-n (Step 2). The corresponding carbapenem derivative (1 equiv.) was treated with Pt/C 10% wt. (1 equiv. mass.) in a mixture of THF:triethylammonium bicarbonate buffer (1 M, pH = 8.5) (2:1) and the reaction mixture was hydrogenated under 3.5 bars for 2 hours at room temperature using the PARR apparatus.

The crude mixture was filtered through a celite pack to remove the catalyst. THF was removed under reduced pressure, and H2O was added. The crude mixture was purified by HPLC using a solvent system consisting of H2O and acetonitrile (linear gradient, 0 to 100% over 45 minutes) and the selected fractions were collected and lyophilized to afford the final peptido-carbapenem derivatives.


Sulaiman Khan
email: sulaiman3355@gmail.com

David Newport
email: david.newport@ul.ie

Stéphane Le Calvé
email: slecalve@unistra.fr

A Sensitive and Portable Deep-UV Absorbance Detector with a Microliter Gas Cell Compatible with Micro GC

Keywords: gas sensor, VOCs, optical sensor, deep-UV LED, photodiodes, analytical device, air quality, UV spectrometry, hollow core waveguide, fiber optic sensor

Deep-UV absorption spectrometry for detection of toxic airborne gases, for instance, Benzene, Toluene, Ethylbenzene, and Xylenes (BTEX) has drawn considerable attention owing to its high sensitivity and reliability. However, the development of a deep-UV absorbance detector having good sensitivity, portability, and a low-volume gas cell with applicability for a micro Gas Chromatography (µGC) is challenging. Herein we present a novel, self-referenced, and portable deep-UV absorbance detector with a microliter (275 µL) gas cell having minimal dead volume. It has excellent compatibility with µGC for detection of individual BTEX components in a mixed sample at a sub-ppm level. The design consists of the latest, portable, and cost-effective optical and electronic components, i.e., deep-UV LED, hollow-core waveguide, and photodiodes. The detector directly measures the absorbance values in volts using an integrated circuit with a log-ratio amplifier. The prototype was tested with direct injection of toluene-N 2 (1.5 ppm to 50 ppm) and good linearity (R 2 = 0.99) with a limit of detection of 196 ppb was obtained. The absorbance detector with µGC setup was tested with a BTEX mixture in N 2 at different GC column temperatures. All the BTEX species were sequentially separated and detected with an individual peak for a concentration range of 2.5 ppm to 10 ppm.

Introduction

Indoor air quality has attracted significant attention for improving the comfort and health of indoor occupants. Poor indoor air quality is becoming a serious health concern as it can lead to health problems, impaired learning, and reduced productivity [START_REF]Introduction to Indoor Air Quality Indoor Air Quality (IAQ)[END_REF]. Indoor air mainly contains Volatile Organic Compounds (VOCs), airborne particles, microorganisms, household odours, and gases. VOCs constitute a considerable portion of indoor airborne pollutants [START_REF] Cincinelli | Indoor Air Quality and Health[END_REF][START_REF] Koppmann | Chemistry of Volatile Organic Compounds in the Atmosphere[END_REF][START_REF] Noziere | The Molecular Identification of Organic Compounds in the Atmosphere: State of the Art and Challenges[END_REF]. Among these VOCs, Benzene, Toluene, Ethylbenzene, and Xylenes (BTEX) pose an alarming threat to human health. They are aromatic hydrocarbons and its common indoor emission sources are coal burning, cigarette smoking, combustion, use of cleaning products, laser printing, floor adhesives, paint, wood panelling, and evaporation of fuel from an adjoining garage [START_REF] Wallace | Major sources of benzene exposure[END_REF][START_REF] Ilgen | Aromatic hydrocarbons in the atmospheric environment: Part I. Indoor versus outdoor sources, the influence of traffic[END_REF][START_REF] Ayoko | Volatile Organic Compounds in Indoor Environments[END_REF]. The infiltration of outside air can also contribute to high BTEX levels, particularly near industrial sources or high road traffic. Long-term exposure to airborne BTEX causes fatigue, respiratory, kidney, nervous system, mental, and cardiovascular-related problems [START_REF] Who | Exposure to Benzene: A Major Public Health Concern[END_REF][START_REF] Baan | A review of human carcinogens-Part F: Chemical agents and related occupations[END_REF][START_REF] Patnaik | A Comprehensive Guide to the Hazardous Properties of Chemical Substances[END_REF][START_REF] Kandyala | Xylene: An overview of its health hazards and preventive measures[END_REF]. Among BTEX, benzene is carcinogenic, as classified by the International Agency for Research on Cancer [START_REF] Pyatt | A review of the potential association between childhood leukemia and benzene[END_REF]. There are strict regulations about the exposure limit of BTEX in indoor spaces. For instance, the National Institute of Occupational Safety and Health (NIOSH) and the Occupational Safety and Health Administration (OSHA) have set an exposure limit of 0.1 ppm and 1 ppm, respectively, for benzene in indoor spaces. For toluene, the exposure limit established by NIOSH and OSHA is 100 ppm and 200 ppm, respectively [START_REF] Barson | NIOSH-Pocket Guide to Chemical Hazards[END_REF][START_REF] Khan | A Review of Optical Interferometry Techniques for VOC Detection[END_REF].

A highly sensitive gas detector having excellent reliability and ease of integration for instance with GC is therefore needed for monitoring of BTEX in indoor spaces. Sensors coupled with micro gas chromatography (µGC), usually called BTEX analysers, can detect each component in the BTEX mixture [START_REF] Allouch | miniature, fast and high sensitive real-time analyzers: BTEX detection[END_REF][START_REF] Khan | Low-volume PEEK gas cell for BTEX detection using portable deep-UV absorption spectrophotometry[END_REF][START_REF] Nasreddine | Portable novel micro-device for BTEX real-time monitoring: Assessment during a field campaign in a low consumption energy junior high school classroom[END_REF]. Optical gas sensors have demonstrated high sensitivity and selectivity for detection of different airborne gases. They have a quick time response with minimal drift and poisoning issues in comparison to other sensing methods for instance electrochemical sensors and metal-oxide semiconductor gas sensors [START_REF] Khan | A Review of Optical Interferometry Techniques for VOC Detection[END_REF]. BTEX molecules absorb strongly in the wavelength range of 240 nm to 275 nm [START_REF] Gordon | The HITRAN2016 molecular spectroscopic database[END_REF]. Thus, absorption spectrometry offers a sensitive, rapid, and non-destructive approach for airborne BTEX detection. This optical detection method is self-referenced and reliable with a minimal cross-response to other molecules. Such optical sensors are based on directly measuring the inherent physical property of a molecule, i.e., absorbance of a molecule at a specific wavelength, which effectively acts as a fingerprint. Recent advancements in optoelectronics and optofluidics have led to the development of LEDs, photodiodes, and hollow-core waveguides (HCW), for the UV-range which can be exploited to develop a portable deep UV absorbance detector.

LEDs provide a portable and quasi-monochromatic UV emission source with good spatial stability, low power input, and lower cost compared to conventional UV emission sources, like mercury lamps. Aside from their cost and bulk size, it is not possible to achieve the target wavelength range for BTEX with Deuterium and Xenon lamps without costly optical filters. For the detection part, photodiodes offer a portable and an efficient UV photodetector: They exhibit good photocurrent linearity, visible-blindness, low-dark current, high quantum efficiency, low input power and noise, portability, and low cost [START_REF] Macka | Light-Emitting Diodes for Analytical Chemistry[END_REF]. In addition to a UV source and photodetector, a gas cell with a high length-to-volume is required to have a compact instrument compatible with GC for the detection of BTEX mixture at ppb or sub-ppm level. In view of these constraints, Hollow-Core Waveguides (HCW) is an excellent candidate that can be applied as a miniature gas cell with an efficient radiation transmission. HCWs transmit UV radiation in a leaky-mode through the reflection of the emitted radiation inside the co-axial hollow core. Aluminium is considered to be an efficient coating material for deep-UV radiation compared to other metals, for instance silver, and it has good chemical compatibility with airborne BTEX molecules [START_REF] Khan | Gas Detection Using Portable Deep-UV Absorption Spectrophotometry: A Review[END_REF].

BTEX components have overlapping spectra and the detection of individual component with high sensitivity in BTEX mixture is challenging. However, an absorbance detector compatible with µGC can separate, identify, and detect each molecule of BTEX mixture. Such a photometer needs a gas cell with a microliter inner volume and larger length having minimum dead volume to detect each BTEX molecule with an individual peak. Previous research on BTEX detection was primarily focussed on the sensitivity of the sensor and mostly an individual BTEX molecule was tested. Eckhardt et al. [START_REF] Eckhardt | Fibre-optic UV systems for gas and vapour analysis[END_REF][START_REF] Eckhardt | Fiber-optic based gas sensing in the UV region[END_REF] developed a detector with an aluminium-coated HCW (length, 1 m and diameter, 1 mm) as a gas cell, deuterium lamp (30 W) as UV source, and CCD-based spectrometer as a UV detector. The setup was coupled with GC and tested for detection of aromatics compounds like ethylbenzene and bromobenzene. Its main limitations are the high volume of the gas cell (785 µL) and the overall size of the spectrophotometer (emission source and detector). Bui et al. [START_REF] Bui | A deep-UV light-emitting diode-based absorption detector for benzene, toluene, ethylbenzene, and the xylene compounds[END_REF] employed deep-UV LED (λ peak = 260 nm) and photodiodes for the direct detection of BTEX with good sensitivity (152 µA•U-ppm -1 ; limit of detection of 0.66 ppm for toluene). An aluminium-based gas cell (length, 40 cm and inner diameter, 2 mm) was used with an inner volume of 1256 µL. The higher inner volume of the gas cell, which limits its use for GC application. Khan et al. [START_REF] Khan | Development of a Toluene Detector Based on Deep UV Absorption Spectrophotometry Using Glass and Aluminum Capillary Tube Gas Cells with a LED Source[END_REF] demonstrated a simple toluene detector using UV-LED, HCWs, and spectrometer. Two types of HCWs were investigated: Glass-coated HCW and aluminium-based HCW. A limit of detection of 8.15 ppm was reported with aluminium-coated glass HCW with an inner volume of 275 µL. The sensitivity and high dead volume are its main drawbacks, which limit its application for GC analysis.

In this paper, the development of deep-UV absorbance detector compatible with µGC for detection of the individual BTEX components is reported. The design is based on using deep-UV LED, photodiodes, and HCW with a microliter volume gas cell with negligible dead-volume. The design allows direct measurement of absorbance without mono-chromator and ease of coupling with µGC. To the best of our knowledge, a sensitive and portable deep-UV absorbance detector based on deep-UV LED, photodiodes, and HCW with microliter volume having good compatibility with µGC has not been reported in the literature.

Materials and Methods

Instrumentation

The schematic of the absorbance detector developed is shown in Figure 1. The instrumentation employed in the development of deep-UV detector were fibre-coupled deep-UV LED, glass HCW coated with aluminium, and photodiodes. A fibre-coupled deep-UV LED (Mightex System, Pleasanton, CA, USA) driven by a stable current source (0-100 mA) was used as an UV emission source. The peak wavelength and power range of this LED were 260 nm and 54-80 µW, respectively. The LED was integrated with a fibre-coupled beam splitter (50:50, QP400-025-SR, Ocean Optics, EW Duiven, the Netherlands) to divide the light into two, one acted as a reference while the other served as a test beam passing through the gas cell. An aluminium-based HCW (Doku Engineering, Sendai, Japan), which is composed of a capillary glass tube coated with aluminium, was installed as a gas cell. The HCW of length 34 cm and the inner volume of 275 µL (inner diameter, 1 mm) was fixed in-between the PEEK T-assemblies (IDEX Health & Science, Oak Harbor, WA, USA) to achieve a lowvolume gas cell with a minimal dead volume. PEEK exhibits excellent chemical resistance and high melting point (343 • C) with sufficient mechanical properties for our application, and can be readily machined [START_REF] Schweitzer | Mechanical and Corrosion-Resistant Properties of Plastics and Elastomers[END_REF]. As shown in Figure 1a, custom-made ferrules and nuts were fixed at the end of the HCW to achieve a leakage-free connection. A PEEK T-assembly was micromachined and coupled with the HCW (via ferrules and nuts) and optical fibreconnector to get a leakage free fluidic-optics connection. All the components were fixed tightly to minimise the dead-volume of the assembly. The HCW with T-assemblies was positioned on an optical bench using 3D printed connectors and fixtures. The connectors were designed using Solidworks (Dassault Systèmes, Vélizy-Villacoublay, France) and 3D printed (Ultimaker 3, Geldermalsen, the Netherlands) using ABS (acrylonitrile butadiene styrene). The rendered image of the 3D-printed holder for T-assembly is shown in Figure 2. The holder was designed to accommodate a gas supply tube, HCW, and the SMA connector (head) of optical fibre. It was designed to minimize leakages and to achieve flexibility and robustness in connecting the gas tubing and optical fibre with the HCW. For the desired wavelength range (245 nm to 275 nm), SiC photodiodes (SG01S, Sglux, Berlin, Germany) were employed. The photodiode selected for the test section shown in Figure 1b has a spectral sensitivity of 120 mA/W (active area 0.06 mm 2 ) for a wavelength range of 225 to 285 nm without any external optical filters. A photodiode (S12742, Hamamatsu, Massy, France) with a built-in interference filter was employed as a reference photodiode. The photodiode has a peak wavelength of 254 nm with a spectral The 3D-printed holders were placed at the two ends of the HCW as illustrated in Figure 1a. A silicone sealant was applied at the external surfaces near the inlet and outlet to ensure the sealing of the connectors. The HCW assembly was covered with Styrofoam and aluminium sheet for thermal insulation and protection. The opto-mechanical components (i.e., LED and photodiodes) were aligned on the optical breadboard (MB3090/M, Thorlabs, Maisons-Laffitte, France) using 3D-printed housings. The board was equipped with vibration-isolating feet to avoid baseline shifts due to mechanical movement. The gas cell was thermally insulated to minimise thermal-induced noise. The 3D-printed housing for HCW is shown in Figure S1.

For the desired wavelength range (245 nm to 275 nm), SiC photodiodes (SG01S, Sglux, Berlin, Germany) were employed. The photodiode selected for the test section shown in Figure 1b has a spectral sensitivity of 120 mA/W (active area 0.06 mm 2 ) for a wavelength range of 225 to 285 nm without any external optical filters. A photodiode (S12742, Hamamatsu, Massy, France) with a built-in interference filter was employed as a reference photodiode. The photodiode has a peak wavelength of 254 nm with a spectral response width of 10 nm allowing the deep-UV photometry with negligible influence of stray light. It has spectral sensitivity of 18 mA/W at a wavelength of 254 nm with an active area of 13 mm 2 . For spectral analysis, the emission spectra of the LED was recorded using a mini-spectrometer (C10082CH, Hamamatsu, Hamamatsu City, Japan) to analyse the spectral emission of the LED. Each photodiode was assembled inside a 3D-printed housing, which was fixed on the optical breadboard. The housing was equipped with an SMA connector to align and transmit the beam of UV light from the gas cell onto the active area of photodiode. The setup was optically insulated to avoid the exposure of any stray light.

An integrated circuit was developed for processing the photocurrents from the photodiodes to compute the absorbance in volts, similar as reported by Bui et al. [START_REF] Bui | A deep-UV light-emitting diode-based absorption detector for benzene, toluene, ethylbenzene, and the xylene compounds[END_REF]. A log-ratio amplifier (LOG101 Texas Instruments, RS Components, Beauvais, France) was used to process the photocurrent using logarithmic computation of the two photocurrents. The circuitry was enclosed in a metallic box connected with the ground, as shown in Figure 1b, to minimise the influence of external electromagnetic waves. A low-pass electronic filter was integrated with the amplifier circuit to remove high-frequency noise. The schematic diagram of electronic and optical components is shown in Figure 1a,b. A multimeter (Keithley 2000, Farnell, Limonest, France) was used to record signal from amplifier in volts, and a lab-VIEW based data acquisition system was developed to record the data (time scale 0.1 s) on PC. The data post-processing was performed using Matlab and Spectragryph [START_REF] Menges | Spectragryph-optical spectroscopy software[END_REF].

Gas Mixture Generation and Gas Chromatography Analysis

For direct toluene injection, a toluene-N 2 cylinder (100 ppm ±2%) was used, and different concentrations were generated by diluting it with different N 2 gas flow using mass flow controllers (Bronkhorst, Montigny-lès-Cormeilles, France). The gas generation setup is shown in Figure S2. For GC analysis, a BTEX cylinder (Air Products, Schiltigheim, France) with a total concentration of 60 ppm was used as a source of BTEX injection. Each component of BTEX mixture constitutes a concentration of 10 ppm ±2%. Similarly, to the generation of gaseous toluene mixture, the dilution bench was used to generate a BTEX-N 2 mixture with a total concentration of 30 ppm and 15 ppm. A pressure controller (IQP600, 0-10 bar ±5% Bronkhorst, Montigny-lès-Cormeilles, France) was installed to maintain the supply of carrier gas inside the GC setup.

A PDMS based GC column was selected due to its polarity and molecular structure, which is compatible with BTEX molecules. The methyl-groups (-CH 3 ) in the molecular structure of PDMS are non-polar and can be replaced with other groups to tune the polarity for the desired application. PDMS (94%) and cyanopropylphenyl (6%) was used for coating the column. The column with a length of 20 m (Inner diameter 180 µm, RXi-624Sil MS) was coiled into a small circle with a diameter of 7.5 cm. The coiled column was placed inside an aluminium plate (10 cm × 10 cm × 1 cm) for uniform heat transfer using an electrical resistor with a thermocouple placed under the column. The column was thermally insulated, and its temperature was regulated via a controller (CAL3300, Farnell, Limonest, France). The gas flow rate inside the µGC during the different phases of chromatography was controlled using a solenoid 6-ports value (MTV-6LL-N32UF-1, Takasago Fluidic Systems, Nagoya, Japan). The 6-ports value was coupled with a sampling loop of volume 200 µL (PEEK Interchim, Montluçon, France). The µGC setup with the absorbance detector is shown in Figure 3.

Results and Discussion

The absorbance detector is designed to directly measure the absorbance of the gas components as a voltage, which is recorded with a multimeter. The emitted UV radiation of the LED is sensitive towards the input current and temperature of the LED. To minimize the fluctuation of the signal, a referencing technique is applied. The UV light emitted from the LED is split into two beams using a fibre-coupled beam splitter, one beam Io acts as a reference, while the other serves as a measurement beam I, passes through the gas cell, as represented in Figure 1b. The two intensities (i.e., Io and I) are recorded using photodiodes, which generate photocurrents io and i, respectively. The photocurrents generated are proportional to the intensity of the incident UV light. A log-ratio amplifier processes the two photocurrents to compute the absorbance by taking a logarithmic ratio of io and i, and gives a single output in volts. The output voltage represents the absorbance according to the Beer-Lambert equation which can be expressed as

A = log I o I (1)
A v = log 𝑖 𝑜 𝑖 (2)

Results and Discussion

The absorbance detector is designed to directly measure the absorbance of the gas components as a voltage, which is recorded with a multimeter. The emitted UV radiation of the LED is sensitive towards the input current and temperature of the LED. To minimize the fluctuation of the signal, a referencing technique is applied. The UV light emitted from the LED is split into two beams using a fibre-coupled beam splitter, one beam I o acts as a reference, while the other serves as a measurement beam I, passes through the gas cell, as represented in Figure 1b. The two intensities (i.e., I o and I) are recorded using photodiodes, which generate photocurrents i o and i, respectively. The photocurrents generated are proportional to the intensity of the incident UV light. A log-ratio amplifier processes the two photocurrents to compute the absorbance by taking a logarithmic ratio of i o and i, and gives a single output in volts. The output voltage represents the absorbance according to the Beer-Lambert equation which can be expressed as

A = log I o I (1)
A v = log i o i (2)
where A v is the absorbance in volts, i o and i represents the photocurrents from the reference photodiode and test photodiode, respectively.

The absorption spectrum of the target BTEX molecules, were initially investigated and compared with the emission spectrum of the LED. The absorption data of the molecules were collected from high-resolution transmission molecular absorption database (HITRAN) [START_REF] Gordon | The HITRAN2016 molecular spectroscopic database[END_REF] and were mapped with the emission spectrum of the LED as shown in Figure 4a. The emission spectrum of the LED was recorded using the mini-spectrometer (C10082CH, Hamamatsu, Hamamatsu city, Japan) without passing through the gas cell. The LED spectrum covers a significant portion of the absorption spectra of the BTEX components for a wavelength range of 245 nm to 275 nm with a peak at 260 nm. The maximum peak absorption cross-sections σ of benzene, toluene, ethylbenzene, m-xylene, o-xylene, and p-xylene with the corresponding peak wavelength are summarized in Table 1. For comparison, the integrated absorbance for wavelength range (245 nm to 275 nm) was calculated by using area integral of the absorption spectrum as given in Figure 4b. This data is important during detection of the BTEX mixture, as the detection of the components is dictated by their corresponding absorbance. p-xylene shows the highest absorption, while benzene shows the lowest absorption, implying that detection of p-xylene is easier compared to other components.

Detection of Toluene

The absorbance detector was first tested with a direct injection of different concentrations of toluene in N2. The experiments were carried out by first injecting pure N2, followed by injection of the toluene-N2 mixture for 5 min. When the toluene molecules were injected into the gas cell, the output voltage recorded increased due to the absorbance of

Detection of Toluene

The absorbance detector was first tested with a direct injection of different concentrations of toluene in N 2 . The experiments were carried out by first injecting pure N 2 , followed by injection of the toluene-N 2 mixture for 5 min. When the toluene molecules were injected into the gas cell, the output voltage recorded increased due to the absorbance of toluene molecules. For instance, the recorded signal for the toluene-N 2 mixture of 5 ppm and 20 ppm is shown in Figure 5. Each concentration was injected three times with pure N 2 injections in-between. A significant rise in the signal was observed, as the toluene concentrations were injected into the cell. High spikes were observed in the absorbance signal at the instant when the toluene cylinder value was turned ON. The gas dynamics inside the capillary tube at the instant of valve opening could be the reason for these instant spikes. No such spikes were observed in case of BTEX detection with µGC, which is discussed in the following section. The experimental device was then tested with direct injection of a toluene-N2 concentration of 1.5 ppm to 50 ppm. A calibration curve for concentrations (1.5 ppm to 50 ppm) of toluene is shown in Figure 6. Each data point on the calibration curve represents an average of three observations while each observation is an average of absorbance values for five mins. The design demonstrated good linearity (R 2 = 0.99) for the investigated concentration range 1.5-50 ppm with a sensitivity of 1.76 × 10 -4 A•U-ppm -1 . A limit of detection of 196 ppb was derived from the curve by using a concentration corresponding to a signal with value three times higher than the baseline noise. The baseline noise was calculated by using a standard deviation of the blank obtained with pure nitrogen flow for a time duration of 250 s (Figure S3) and repeated three times. The experimental device was then tested with direct injection of a toluene-N 2 concentration of 1.5 ppm to 50 ppm. A calibration curve for concentrations (1.5 ppm to 50 ppm) of toluene is shown in Figure 6. Each data point on the calibration curve represents an average of three observations while each observation is an average of absorbance values for five mins. The design demonstrated good linearity (R 2 = 0.99) for the investigated concentration range 1.5-50 ppm with a sensitivity of 1.76 × 10 -4 A•U-ppm -1 . A limit of detection of 196 ppb was derived from the curve by using a concentration corresponding to a signal with value three times higher than the baseline noise. The baseline noise was calculated by using a standard deviation of the blank obtained with pure nitrogen flow for a time duration of 250 s (Figure S3) and repeated three times.

The experimental device was then tested with direct injection of a toluene-N2 concentration of 1.5 ppm to 50 ppm. A calibration curve for concentrations (1.5 ppm to 50 ppm) of toluene is shown in Figure 6. Each data point on the calibration curve represents an average of three observations while each observation is an average of absorbance values for five mins. The design demonstrated good linearity (R 2 = 0.99) for the investigated concentration range 1.5-50 ppm with a sensitivity of 1.76 × 10 -4 A•U-ppm -1 . A limit of detection of 196 ppb was derived from the curve by using a concentration corresponding to a signal with value three times higher than the baseline noise. The baseline noise was calculated by using a standard deviation of the blank obtained with pure nitrogen flow for a time duration of 250 s (Figure S3) and repeated three times.

Detection of BTEX with µGC

The detector was then tested for detection of BTEX mixtures by coupling it with the µGC setup developed by our research group. The µGC process for separation and detection of the BTEX mixture was carried out in two modes, i.e., the sampling mode and analysis mode, as described in the Section 2.1. The separation and detection of BTEX were conducted in the analysis mode. In the sampling mode, the BTEX species were sampled into a loop of fixed volume (200 µL) at a flow rate of 30-50 mL/min (P = 1 bar, T = 25 • C) for 15 s. The carrier gas N 2 was passing through the column and detector at a flow rate of 2.45 mL/min. The transition from sampling mode to analysis mode was carried out with the 6-ports valve. During the analysis mode, the 6-ports valve was turned ON and the BTEX components were injected into the µGC. The separated species were then injected into the absorbance detector as shown in Figure 3. In the GC analysis, the 6-ports valve switches very rapidly between sampling and analysis modes, whereas the pressure was maintained constant by a forward pressure controller. There was no valve opening/closing involved, therefore no spikes were observed at the instant of injection of BTEX.

A total BTEX-N 2 mixture of 60 ppm with each compound of 10 ppm was injected into GC. The µGC column's temperature was maintained at 60 • C. A chromatogram of 700 s was recorded in which all the peaks were separated, corresponding to benzene, toluene, ethylbenzene, (m-,p-) xylenes, and o-xylene in temporal sequence, as shown in Figure 7 4b). The device was then tested for total BTEX-N 2 mixture of 30 ppm and 15 ppm, and the concentration of the individual molecule was 5 ppm (orange line) and 2.5 ppm (yellow line), respectively. The chromatograms for the separated BTEX species are shown in Figure 7. All the BTEX components were detected for all three concentrations injected. However, a weak signal was obtained for 2.5 ppm with no clear signal for benzene and ethylbenzene. This is due to the relatively lower absorption coefficient of benzene and ethylbenzene. 9a). At the highest temperature, i.e., 80 • C, a chromatogram was obtained in a shorter time with lower values for peaks resolution. The peak for ethylbenzene is not discernible for the case of 80 • C, as observed in Figure 9c. Therefore, the optimal temperature was found to be 60 • C, as already reported by Nasreddine et al. [START_REF] Nasreddine | Development of a novel portable miniaturized GC for near real-time low level detection of BTEX[END_REF], where they used a photo-ionisation detection module. The peaks-resolution values for BTEX components at the three studied temperatures are summarized in Table 2. The degree of separation between the two peaks (a and b) was quantified by using a resolution, R, as given by [START_REF] Harvey | Modern Analytical Chemistry[END_REF],

R = t b r -t a r 0.5 (w b -w a ) (3)
where t r and w represent the retention time and baseline width (full width at half maximum-FWHM), respectively. At lower temperature (i.e., 40 • C), peak resolutions have higher values (about two times higher) compared to those obtained with the column temperature of 60 • C. During testing all the gases were flushed into the systems from cylinders with a negligible level of humidity. Although the detector signal is not affected by humidity in the air. However, humidity could absorb into the reflective surface of the HCW and decrease the transmission of UV radiation with time. Nafion membrane at the inlet or quartz lining of the aluminium coating are usually used to avoid the negative effects of humidity on the sensing system [START_REF] Khan | Gas Detection Using Portable Deep-UV Absorption Spectrophotometry: A Review[END_REF].

Conclusions

A novel, self-referenced deep-UV absorbance detector was developed for the detection of BTEX components. The absorbance detector composed of portable devices: UV-LED, HCW as a gas cell, and photodiodes. The detector directly measures the absorbance values in volts. The design is based on using a log-ratio amplifier, which used the photocurrents from a reference and measurement photodiodes to compute the absorbance in

15 Figure 1 .

 151 Figure 1. (a) Schematic of ultraviolet (UV) absorbance detector: Gas cell with gas and optics connectors. (b) Schematic for measuring the absorbance in volts. LabVIEW based data acquisition (DAQ) system was used to record the data.

Figure 1 .

 1 Figure 1. (a) Schematic of ultraviolet (UV) absorbance detector: Gas cell with gas and optics connectors. (b) Schematic for measuring the absorbance in volts. LabVIEW based data acquisition (DAQ) system was used to record the data.

Figure 1 .

 1 Figure 1. (a) Schematic of ultraviolet (UV) absorbance detector: Gas cell with gas and optics connectors. (b) Schematic for measuring the absorbance in volts. LabVIEW based data acquisition (DAQ) system was used to record the data.

Figure 2 .

 2 Figure 2. The rendered image of the 3D-printed holder for the two ends of the absorbance detector. The T-assembly with connected optical fibre, gas tube, and hollow-core waveguide (HCW) was placed inside the holder, and covered by the top lid to minimise any misalignment and vibrations. The whole assembly was then fixed on the optical bread board with screws.

Figure 2 .

 2 Figure 2. The rendered image of the 3D-printed holder for the two ends of the absorbance detector. The T-assembly with connected optical fibre, gas tube, and hollow-core waveguide (HCW) was placed inside the holder, and covered by the top lid to minimise any misalignment and vibrations. The whole assembly was then fixed on the optical bread board with screws.

Figure 3 .

 3 Figure 3. Schematic of the μGC setup with the absorbance detector. Sampling mode, i.e., 6-ports value-OFF (top) and the analysis mode, i.e., 6-ports value-ON (bottom) for GC.

Figure 3 .

 3 Figure 3. Schematic of the µGC setup with the absorbance detector. Sampling mode, i.e., 6-ports value-OFF (top) and the analysis mode, i.e., 6-ports value-ON (bottom) for GC.

Chemosensors 2021, 9 , 15 Figure 4 .

 9154 Figure 4. (a) LEDs emission spectrum mapped on the absorption spectrum of benzene, toluene and m-xylene, p-xylene and o-xylene. Data was sourced from HITRAN [18] and then plotted according to the scale. Each spectrum is normalized by its maximum peak value. (b) The area integral of BTEX components for a wavelength range 245-275 nm.

Figure 4 .

 4 Figure 4. (a) LEDs emission spectrum mapped on the absorption spectrum of benzene, toluene and m-xylene, p-xylene and o-xylene. Data was sourced from HITRAN [18] and then plotted according to the scale. Each spectrum is normalized by its maximum peak value. (b) The area integral of BTEX components for a wavelength range 245-275 nm.

  Chemosensors 2021, 9, x FOR PEER REVIEW 9 of 15spikes. No such spikes were observed in case of BTEX detection with μGC, which is discussed in the following section.

Figure 5 .

 5 Figure 5. Absorbance signal in milli-volts recorded for the toluene-N2 mixture of 5 ppm and 20 ppm. Pure nitrogen was injected in-between the different gaseous toluene injections.

Figure 5 .

 5 Figure 5. Absorbance signal in milli-volts recorded for the toluene-N 2 mixture of 5 ppm and 20 ppm. Pure nitrogen was injected in-between the different gaseous toluene injections.

Figure 6 .

 6 Figure 6. Calibration curve representing the absorbance vs. the gaseous toluene concentration in the range 1.5-50 ppm. The error bars represent the standard deviation of absorbance for three experiments.

  (red line). Each peak represents an individual BTEX molecule. The elution sequence of BTEX molecule is performed according to the boiling points of BTEX components. The boiling points of m-xylene and p-xylene are very close (i.e., 139.1 • C and 138.4 • C, respectively) so that they exhibit coelution. (m-,p-) xylenes have the highest peak followed by o-xylene, toluene, ethylbenzene, and benzene, which is in accordance with the absorbance value of the components for the wavelength range of 245 nm to 275 nm (see Figure

Figure 7 .

 7 Figure 7. Chromatograms obtained for the toluene-N2 concentration of 10 ppm, 5 ppm, and 2.5 ppm at column temperature 60 °C .

Figure 8 .

 8 Figure 8. Peak's area of different concentration (i.e., 10 ppm, 5 ppm, and 2.5 ppm) of BTEX-N2 at column temperature of 60 °C . B, T, E, X1, and X2 represent benzene, toluene, ethylbenzene, (m-,p-) xylenes, and o-xylene, respectively. The error bar represents the standard deviation of absorbance for three experiments. Furthermore, experiments were conducted at a different column temperature of 40 °C , 60 °C , and 80 °C as illustrated in Figure 9. At the lowest temperature, i.e., 40 °C , all the BTEX were distinctively separated at a cost of extended time for the chromatogram (see

Figure 7 .

 7 Figure 7. Chromatograms obtained for the toluene-N 2 concentration of 10 ppm, 5 ppm, and 2.5 ppm at column temperature 60 • C. An area of each peak was calculated for the BTEX molecule and plotted for the three concentrations (10 ppm, 5 ppm, 2.5 ppm) as shown in Figure 8. The detector demonstrated good linearity for all the BTEX components with R 2 ~0.99. It should be noted that only three data points were used for calibration due to the limitation of the BTEX cylinder and MFCs employed in the concentration generation setup. A sensitivity of 1.91 × 10 -4 A•U-ppm -1 , 6.55 × 10 -4 A•U-ppm -1 , 1.34 × 10 -4 A•U-ppm -1 , 9.58 × 10 -4 A•U-ppm -1 , and 5.70 × 10 -4 A•U-ppm -1 was calculated for benzene, toluene, ethylbenzene, (m-,p-) xylenes, and o-xylene, respectively. The pattern of sensitivities is according to the absorbance cross-section values of the species.

Figure 7 .

 7 Figure 7. Chromatograms obtained for the toluene-N2 concentration of 10 ppm, 5 ppm, and 2.5 ppm at column temperature 60 °C .

Figure 8 .

 8 Figure 8. Peak's area of different concentration (i.e., 10 ppm, 5 ppm, and 2.5 ppm) of BTEX-N2 at column temperature of 60 °C . B, T, E, X1, and X2 represent benzene, toluene, ethylbenzene, (m-,p-) xylenes, and o-xylene, respectively. The error bar represents the standard deviation of absorbance for three experiments.

Figure 8 .

 8 Figure 8. Peak's area of different concentration (i.e., 10 ppm, 5 ppm, and 2.5 ppm) of BTEX-N 2 at column temperature of 60 • C. B, T, E, X1, and X2 represent benzene, toluene, ethylbenzene, (m-,p-) xylenes, and o-xylene, respectively. The error bar represents the standard deviation of absorbance for three experiments. Furthermore, experiments were conducted at a different column temperature of 40 • C, 60 • C, and 80 • C as illustrated in Figure 9. At the lowest temperature, i.e., 40 • C, all the BTEX were distinctively separated at a cost of extended time for the chromatogram (see Figure9a). At the highest temperature, i.e., 80 • C, a chromatogram was obtained in a shorter time with lower values for peaks resolution. The peak for ethylbenzene is not discernible for the case of 80 • C, as observed in Figure9c. Therefore, the optimal temperature was found to be 60 • C, as already reported by Nasreddine et al.[START_REF] Nasreddine | Development of a novel portable miniaturized GC for near real-time low level detection of BTEX[END_REF], where they used a photo-ionisation detection module. The peaks-resolution values for BTEX components at the three studied temperatures are summarized in Table2. The degree of separation between the two peaks (a and b) was quantified by using a resolution, R, as given by[START_REF] Harvey | Modern Analytical Chemistry[END_REF],

Figure 9 .

 9 Figure 9. Chromatograms obtained with the μGC coupled to our spectrometer for 10 ppm of BTEX components at column temperatures (a) 40 °C, (b) 60 °C, and (c) 80 °C.

Figure 9 .

 9 Figure 9. Chromatograms obtained with the µGC coupled to our spectrometer for 10 ppm of BTEX components at column temperatures (a) 40 • C, (b) 60 • C, and (c) 80 • C.

Table 2 .

 2 Peak-resolution values for BTEX components at different temperatures.

Table 1 .

 1 Maximum peak absorption cross-section σ of Benzene, Toluene, Ethylbenzene, and Xylenes (BTEX) components at the corresponding peak wavelengths[START_REF] Gordon | The HITRAN2016 molecular spectroscopic database[END_REF].

	Molecule	Peak Wavelength (nm)	Maximum Peak Absorption Cross-Section σ × 10 -18 (cm 2 -Molecule -1 )
	Benzene	253.0	7.73
	Toluene	266.8	1.21
	m-xylene	270.6	1.15
	o-xylene	259.7	0.77
	p-xylene	272.3	3.07

Time (1st-Last Peak) sec

	40	9.35	17.88	2.65	6.75	1038
	60	4.37	9.76	1.5	2.85	450
	80	2.06	3.57 a		1.06	220

a Ethyl benzene coeluted with m-xylene and p-xylene.

Acknowledgments:

The authors appreciate the technical support from Peter C. Hauser, Department of Chemistry, University of Basel Switzerland, in the development of electronic circuitry for data acquisition.

Funding: This work was funded by European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Innovative Training Network-MIGRATE (Miniaturized Gas flow foR Applications with Enhanced Thermal Effects), grant agreement No. 643095 [H2020-MSCA-ITN-2014]. This work has also received funding from the Clean Sky 2 Joint Undertaking under the European Union's Horizon 2020 research and innovation program under grant agreement No 687014 (MACAO Project). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable.

Data Availability Statement:

The data presented in this study are available on request from the corresponding author. The data are not publicly available because the authors want to keep priority for conference presentations.

Conclusions

A novel, self-referenced deep-UV absorbance detector was developed for the detection of BTEX components. The absorbance detector composed of portable devices: UV-LED, HCW as a gas cell, and photodiodes. The detector directly measures the absorbance values in volts. The design is based on using a log-ratio amplifier, which used the photocurrents from a reference and measurement photodiodes to compute the absorbance in volts according to Beer-Lambert law. The design was tested with direct injection of toluene and with a mixture of BTEX species. For toluene, the detector response increases linearly with the gaseous toluene concentration in the range 1.5-50 ppm, i.e., toluene levels consistent with the exposure limits of 100-200 ppm given by NIOSH and OSHA. A corresponding sensitivity and limit of detection of 1.76 × 10 -4 A•U-ppm -1 and 196 ppb, respectively, were obtained for toluene. The absorbance detector was also coupled with µGC set up, enabling the separation of BTEX components prior to their detection by UV absorbance detector. All the BTEX components were separated and detected with an individual peak correspond to each BTEX molecule for a concentration range of 10 ppm to 2.5 ppm. The design was also tested at a different column temperature of 40, 60, and 80 • C. The proposed detection device has low volume with minimal dead-volume and directly measures the absorbance with a stable signal. The design has tremendous potential to be employed for the chromatographic studies of different analytes in media like gas or liquid.

Supplementary Materials:

The following are available online at https://www.mdpi.com/article/10 .3390/chemosensors9040063/s1, Figure S1: The rendered image of the 3D printed housing for HCW. Multiple housing/holders were 3D printed and installed on the optical breadboard to support and align the HCW; Figure S2

Conflicts of Interest:

The authors declare no conflict of interest.


Tristan Berr

Jeanne Naudet

Cynthia Lagourgue

Kiara Vuibert

Karen Bourgeois

Éric Vidal

Plastic ingestion by seabirds in New Caledonia, South Pacific

Keywords:

The accumulation of plastic pollutants in marine environments has many adverse effects on wildlife. In particular, marine predators are often exposed to accidental plastic ingestion, that may negatively affect survival due to the concentration of debris in the digestive tract. Among the species most vulnerable to plastic ingestion, seabirds are of major interest for conservation because of their wide foraging areas, long generation time and extended lifespan. We analysed stomach contents of 90 seabird specimens from 12 different species collected in New Caledonia to assess the local prevalence of plastic ingestion. Overall, we found plastic debris in 14.4% of sampled individuals, exclusively in procellariids: Gould's Petrel (41.2%, highest incidence), Tahiti Petrel (33.3%) and Wedge-tailed Shearwater (7.7%). To our knowledge, this study is the first characterization of plastic ingestion in seabirds from New Caledonia and our results show an overall lower ingestion prevalence compared to other assessments in the tropical Pacific.

The ever-growing concentration of plastic waste in marine environments is a major concern for wildlife [START_REF] Derraik | The pollution of the marine environment by plastic debris: a review[END_REF][START_REF] Barnes | Accumulation and fragmentation of plastic debris in global environments[END_REF][START_REF] Eriksen | Plastic pollution in the world's oceans: more than 5 trillion plastic pieces weighing over 250,000 tons afloat at sea[END_REF][START_REF] Gall | The impact of debris on marine life[END_REF]. Within the marine megafauna, seabirds are severely affected by the presence of debris in shallow waters [START_REF] Ryan | The incidence and characteristics of plastic particles ingested by seabirds[END_REF][START_REF] Wilcox | Threat of plastic pollution to seabirds is global, pervasive, and increasing[END_REF][START_REF] Tavares | Incidence of marine debris in seabirds feeding at different water depths[END_REF]. Their surface-feeding and resting behaviours make them highly susceptible to entanglement [START_REF] Votier | The use of plastic debris as nesting material by a colonial seabird and associated entanglement mortality[END_REF] and ingestion [START_REF] Gregory | Environmental implications of plastic debris in marine settings-entanglement, ingestion, smothering, hangers-on, hitch-hiking and alien invasions[END_REF][START_REF] Roman | Anthropogenic debris ingestion by avifauna in eastern Australia[END_REF], thus increasing mortality risks [START_REF] Wilcox | Threat of plastic pollution to seabirds is global, pervasive, and increasing[END_REF]Roman et al., 2019b). In particular, plastic ingestion has multiple deleterious effects: obstruction or damage of the intestinal tract [START_REF] Kühn | Deleterious effects of litter on marine life[END_REF], dehydration and reduced food intake [START_REF] Auman | Plastic ingestion by Laysan Albatross chicks on Sand Island, Midway Atoll, in 1994 and 1995[END_REF] and/or accumulation of plastic-derived chemicals in the body [START_REF] Tanaka | Accumulation of plastic-derived chemicals in tissues of seabirds ingesting marine plastics[END_REF][START_REF] Tanaka | Facilitated leaching of additive-derived PBDEs from plastic by seabirds' stomach oil and accumulation in tissues[END_REF]. Incidence, size and colour of ingested particles strongly differ across seabird species and geographic regions [START_REF] Ryan | The incidence and characteristics of plastic particles ingested by seabirds[END_REF][START_REF] Ainley | The incidence of plastic in the diets of pelagic seabirds in the Eastern Equatorial Pacific region[END_REF][START_REF] Roman | Anthropogenic debris ingestion by avifauna in eastern Australia[END_REF]. This is thought to derive mostly from local variations of plastic abundance [START_REF] Van Franeker | Seabirds, gyres and global trends in plastic pollution[END_REF][START_REF] Thiel | Impacts of marine plastic pollution from continental coasts to subtropical gyres-fish, seabirds, and other vertebrates in the SE Pacific[END_REF], diet specificities [START_REF] Azzarello | Marine birds and plastic pollution[END_REF][START_REF] Savoca | Northwest Lagoon, Between Koumac and Yandé, Province Nord, New Caledonia[END_REF][START_REF] Tavares | Incidence of marine debris in seabirds feeding at different water depths[END_REF] and taxonomic traits [START_REF] Sileo | Prevalence and Characteristics of Plastic Ingested by Hawaiian Seabirds[END_REF]Roman et al., 2019a). Multispecies assessments are thus necessary for accurately characterizing ingestion risks in a given location [START_REF] Gilbert | Plastic ingestion in marine and coastal bird species of southeastern Australia[END_REF][START_REF] Basto | Plastic ingestion in aquatic birds in Portugal[END_REF] and developing relevant conservation responses [START_REF] Avery-Gomm | Linking plastic ingestion research with marine wildlife conservation[END_REF].

New Caledonia, South Pacific (21.2°S, 165.9°E) is home to > 25 breeding seabird species [START_REF] Spaggiari | New Caledonian seabirds[END_REF] among which the Tahiti Petrel (Pseudobulweria rostrata, NT), Gould's Petrel (Pterodroma leucoptera, VU) and the Fairy Tern (Sternula nereis, VU) exhibit extinction risks of concern in the IUCN RedList (IUCN, 2019). Its territorial waters also host a significant proportion of the global breeding population of the Wedge-tailed Shearwater (Ardenna pacifica, > 500,000 breeding pairs) (Benoit and Bretagnolle, 2002;[START_REF] Baudat-Franceschi | Seabirds of the Southern Lagoon of New Caledonia; distribution, abundance and threats[END_REF], a species highly vulnerable to plastic ingestion [START_REF] Kain | Plastic ingestion by Newell's (Puffinus newelli) and wedge-tailed shearwaters (Ardenna pacifica) in Hawaii[END_REF][START_REF] Lavers | Ingestion of marine debris by Wedge-tailed Shearwaters (Ardenna pacifica) on Lord Howe Island, Australia during 2005-2018[END_REF]. While local impacts of invasive species are relatively well known [START_REF] Palmas | Feral cats threaten the outstanding endemic fauna of the New Caledonia biodiversity hotspot[END_REF], no information on plastic-associated threats is currently available in this geographic area.

We present here the first characterization of the incidence of plastic ingestion in a set of seabird species breeding in New Caledonia. This work sets a baseline for future monitoring of the extent, variation and severity of plastic-associated risks for local breeding seabird populations.

Individual stomach contents were examined in 90 specimens collected between 2015 and 2018 from the following 12 seabird species: Wedge-tailed Shearwater (n = 52), Gould's Petrel (n = 17), Tahiti Petrel (n = 6), Black Noddy (Anous minutus; n = 4); Roseate Tern T (Sterna dougallii; n = 4), Red-footed booby (Sula sula; n = 1), Silver Gull (Larus novaehollandiae; n = 1), Brown noddy (Anous stolidus; n = 1), Bridled Tern (Onychoprion anaethetus; n = 1), Sooty Tern (Onychoprion fuscatus; n = 1), Fairy Tern (Sternula nereis; n = 1) and Greater Crested Tern (Thalasseus bergii; n = 1). Carcasses were collected opportunistically, either on monitored breeding sites (south-western and north-western lagoons, southern Caledonian chain) or after collision events (roadkill, light-induced misorientation) near urban or industrialized areas. After collection, each specimen received an individual identification number and was stored at -20 °C until dissection.

Dissections were performed following the standard dissection protocol of van Franeker [START_REF] Van Franeker | Litter NSV; marine litter monitoring by northern fulmars (a pilot study[END_REF]. Due to heterogeneous sampling, corpse preservation varied to some extent across specimens and prevented exhaustive characterization of weight and body condition. Specimen age (adult/fledgling/chick) was determined through the examination of the plumage and/or the state of gonad development. The digestive tract (proventriculus, gizzard) was then extracted and its contents were rinsed and filtrated using a 0.5 mm sieve. Remaining fragments were exposed to two consecutive digestive treatments: 30% H 2 O 2 at 60 °C (24 h) and 10% HNO 3 at room temperature (5 min) in order to remove organic matter from the samples [START_REF] Lusher | Sampling, isolating and identifying microplastics ingested by fish and invertebrates[END_REF]. When present, plastic debris were then collected, dried overnight (60 °C) and weighed to the nearest 0.0001 g.

Following the standardized methodology of [START_REF] Provencher | Quantifying ingested debris in marine megafauna: a review and recommendations for standardization[END_REF], plastic items were characterized by type (user/industrial; sheetlike/ threadlike/foamed/fragment/other), maximum length (microplastics: 1-5 mm/mesoplastics: 5-20 mm/macroplastics: 20-100 mm), and colour (off/white-clear/grey-silver/black/blue-purple/green/orange-brown/redpink/yellow). For each species, the percentage Frequency of Occurrence (FO -proportion of individuals having ingested plastic debris) was reported along with Jeffrey's 95% credible interval, and the average number and mass of plastic items per specimen (with standard error and deviation, including zeros) were calculated. These estimates were further assessed for two separate age classes: adult specimens (autonomous feeding) and chicks/fledglings (parental feeding).

All statistical analyses were conducted using R. 3.6.0. Plastic debris were found in stomach contents of 13 birds out of the 90 examined (FO = 14.4%). All three procellariid species had ingested plastic items: Gould's Petrel (average of 1.12 ± 0.37 plastic items per specimen), Tahiti Petrel (0.5 ± 0.42) and Wedge-tailed Shearwater (0.12 ± 0.06) (Tables 1 to 3, mean ± standard error). Individuals from non-procellariid species (n = 15) showed no sign of plastic ingestion. Percentage FO was significantly higher for Gould's Petrel than for Wedge-tailed Shearwater specimens (41.18% vs 7.69% respectively, pairwise proportion test: P < 0.001). FO estimate for Tahiti Petrel yielded an intermediate value (33.33%) but did not differ significantly from the other two species, likely due to small sample size (six specimens only, P > 0.05). Additionally, FO estimates did not vary significantly between chicks/fledglings and adults for all three species (P > 0.05), thus indicating indirect transmission of plastic litter through regurgitation.

All debris were categorized as user plastics, with a majority of fragment and thread-like items (Tables 1 to 3). Microplastics were the most represented size range, followed by meso-and macroplastics (Table 4). Off/white-clear was the most abundant colour among ingested items (53.6% -Table 4). Due to small sample size, no colourbased selection of ingested plastics across species could be firmly demonstrated. Only black and off/white-clear items were retrieved in Tahiti Petrels and Wedge-tailed Shearwaters, while debris found in Gould's Petrels exhibited more diverse colours (off/white-clear/black/ blue-purple/green/yellow).

Risks of plastic ingestion exist for all groups of seabird species but they are most severe in procellariforms [START_REF] Ryan | The incidence and characteristics of plastic particles ingested by seabirds[END_REF][START_REF] Thiel | Impacts of marine plastic pollution from continental coasts to subtropical gyres-fish, seabirds, and other vertebrates in the SE Pacific[END_REF]Roman et al., 2019a). Their high vulnerability toward plastic ingestion is generally associated with their pelagic foraging [START_REF] Van Franeker | Seabirds, gyres and global trends in plastic pollution[END_REF][START_REF] Tavares | Incidence of marine debris in seabirds feeding at different water depths[END_REF], surface feeding behaviour [START_REF] Roman | Anthropogenic debris ingestion by avifauna in eastern Australia[END_REF] and sensitivity to olfactory cues mimicked by decaying debris [START_REF] Savoca | Northwest Lagoon, Between Koumac and Yandé, Province Nord, New Caledonia[END_REF][START_REF] Savoca | The ecology of an olfactory trap[END_REF]. Our findings are coherent with these expectations, as ingested plastics were exclusively found in procellariid species in this study. Intraspecific differences in plastic occurrence (Table 5) may account for differences in diet selectivity and/or existence of separate foraging areas [START_REF] Ainley | The incidence of plastic in the diets of pelagic seabirds in the Eastern Equatorial Pacific region[END_REF]Roman et al., 2019a). We found few previous records of plastic ingestion for Gould's Petrel and Tahiti Petrel in the tropical Pacific [START_REF] Spear | Incidence of plastic in seabirds from the tropical pacific, 1984-1991: relation with distribution of species, sex, age, season, year and body weight[END_REF][START_REF] Roman | Anthropogenic debris ingestion by avifauna in eastern Australia[END_REF] and only one study mentioned the precise localisation of sampled specimens [START_REF] Roman | Anthropogenic debris ingestion by avifauna in eastern Australia[END_REF] (Table 5). In the latter, FO estimates of plastic ingestion were evaluated using only one specimen of Tahiti Petrel (FO = 0%) and two specimens of Gould's Petrel (FO = 100%), thus giving poor statistical support to the comparison of ingestion risks across geographic regions. Conversely, plastic ingestion by Wedgetailed Shearwaters has been more documented in recent years [START_REF] Hutton | Plastic ingestion by Flesh-footed Shearwaters, Puffinus carneipes, and Wedge-tailed Shearwaters, Puffinus pacificus[END_REF][START_REF] Gilbert | Plastic ingestion in marine and coastal bird species of southeastern Australia[END_REF][START_REF] Kain | Plastic ingestion by Newell's (Puffinus newelli) and wedge-tailed shearwaters (Ardenna pacifica) in Hawaii[END_REF][START_REF] Lavers | Ingestion of marine debris by Wedge-tailed Shearwaters (Ardenna pacifica) on Lord Howe Island, Australia during 2005-2018[END_REF]Roman et al., 2019a). For either chick/fledglings or adults, our results showed the lowest FO estimates of plastic ingestion as well as the least number of ingested particles per specimen when compared to previously published data on necropsied birds in the tropical Pacific (Table 5). The risk of plastic ingestion may therefore be smaller for New-Caledonia Shearwaters, likely due to plastic debris being less abundant in neighbouring foraging areas [START_REF] Sebille | A global inventory of small floating plastic debris[END_REF]Roman et al., 2019a).

To our knowledge, only one study mentions positive plastic ingestion records in some of the other, non-procellariid species that were

Table 5

Summary of post-2000 plastic ingestion studies (necropsy-based) in the tropical Pacific area for adults (A) and chicks/fledglings (F) of three procellariid species: Wedge-tailed Shearwater, Gould's Petrel and Tahiti Petrel. [START_REF] Rapp | Community-wide patterns of plastic ingestion in seabirds breeding at French Frigate Shoals, Northwestern Hawaiian Islands[END_REF]. Since most of these sampled taxa are coastal foragers, they are likely less exposed to ingestion risks from pelagic pollution than procellariids [START_REF] Thiel | Impacts of marine plastic pollution from continental coasts to subtropical gyres-fish, seabirds, and other vertebrates in the SE Pacific[END_REF].

It should be noted that post-mortem examination of stomach content does not account for all ingested plastics: debris can be partially regurgitated to chicks [START_REF] Ryan | Intraspecific variation in plastic ingestion by seabirds and the flux of plastic through seabird populations[END_REF][START_REF] Carey | Intergenerational transfer of plastic debris by Short-tailed Shearwaters (Ardenna tenuirostris)[END_REF] or excreted to some extent [START_REF] Ryan | How quickly do albatrosses and petrels digest plastic particles?[END_REF][START_REF] Provencher | Garbage in guano? Microplastic debris found in faecal precursors of seabirds known to ingest plastics[END_REF]. Additionally, the detection of ultrafine/nanoplastics requires more precise detection methods [START_REF] Provencher | Quantifying ingested debris in marine megafauna: a review and recommendations for standardization[END_REF][START_REF] O'hanlon | Seabirds and marine plastic debris in the northeastern Atlantic: a synthesis and recommendations for monitoring and research[END_REF]. Exposure to ingestion risk is thus usually under-evaluated.

Further developments of this work include (1) aggregation of more ingestion data through future specimen collection; (2) analysis of local foraging patterns and (3) assessment of temporal trends of plastic ingestion (long-term study).

Table 1

 1 Occurrence and characteristics of plastics ingested by Gould's Petrel specimens (Pterodroma leucoptera, n = 17), by category. CI: Jeffrey's credible interval. Means, medians & ranges include all sampled individuals.

		Frequency of occurrence (%; 95% CI)	Number of plastic items			Mass of plastic items
			Mean (n; ± sd; ± se)	Median	Range	Mean (g, ± sd, ± se)	Median	Range
	Global	41.18 (20.68; 64.41)	1.12 ( ± 1.54; ± 0.37)	0	0-4	0.0018 ( ± 0.0024; ± 0.0006)	0	0-0.0078
	User	41.18 (20.68; 64.41)	1.12 ( ± 1.54; ± 0.37)	0	0-4	0.0018 ( ± 0.0024; ± 0.0006)	0	0-0.0078
	Industrial	0 (0; 13.55)	0	0	0	0	0	0
	Sheetlike	0 (0; 13.55)	0	0	0	0	0	0
	Threadlike	17.65 (5.23; 40.01)	0.29 ( ± 0.77; ± 0.19)	0	0-3	0.0004 ( ± 0.0011; ± 0.0003)	0	0-0.0037
	Foam	0 (0; 13.55)	0	0	0	0	0	0
	Fragment	35.29 (16.29; 58.86)	0.82 ( ± 1.38; ± 0.33)	0	0-4	0.0014 ( ± 0.0020; ± 0.0005)	0	0-0.0063
	Other	0 (0; 13.55)	0	0	0	0	0	0

Table 3

 3 Occurrence and characteristics of plastics ingested by Wedge-tailed Shearwater specimens (Ardenna pacifica, n = 52), by category. CI: Jeffrey's credible interval. Means, medians & ranges include all sampled individuals. *mass < 0.0001 g.

		Frequency of occurrence (%; 95% CI)	Number of plastic items			Mass of plastic items
			Mean (n; ± sd; ± se)	Median	Range	Mean (n, ± sd, ± se)	Median	Range
	Global	7.69 (2.65; 17.25)	0.12 ( ± 0.43; ± 0.06)	0	0-2	0.0007 ( ± 0.0038; ± 0.0005)	0	0-0.0239
	User	7.69 (2.65; 17.25)	0.12 ( ± 0.43; ± 0.06)	0	0-2	0.0007 ( ± 0.0038; ± 0.0005)	0	0-0.0239
	Industrial	0 (0; 4.69)	0	0	0	0	0	0
	Sheetlike	0 (0; 4.69)	0	0	0	0	0	0
	Threadlike	1.92 (0.21; 8.63)	0.04 ( ± 0.27; ± 0.04)	0	0-2	0*	0	0*
	Foam	0 (0; 4.69)	0	0	0	0	0	0
	Fragment	5.77 (1.65; 14.60)	0.08 ( ± 0.33; ± 0.05)	0	0-2	0.0007 ( ± 0.0038; ± 0.0005)	0	0-0.0239
	Other	0 (0; 4.69)	0	0	0	0	0	0

Table 4

 4 Size and colour characteristics of plastic debris ingested by three procellariid species. : a specimen of Brown Noddy and one of Red-footed Booby were found to have ingested plastic on Tern Island, Hawaii during the 2006-2013 period (out of 18 and 19 necropsied birds respectively)

	Species	Plastic size category (%)			Plastic colour (%)
		Microplastics (1-5 mm)	Mesoplastics (5-20 mm)	Macroplastics (20-100 mm)	Off/White-clear	Black	Blue-purple	Green	Yellow
	All	60.71	32.14	7.14	53.57	14.29	17.86	10.71	3.57
	Gould's Petrel	73.68	21.05	5.26	47.37	5.26	26.32	15.79	5.26
	Tahiti Petrel	0	66.67	33.33	100	0	0	0	0
	Wedge-tailed Shearwater	50	50	0	50	50	0	0	0

sampled

Acknowledgements

We thank all the specimen collectors (volunteers, rangers and nonscientific partners) who contributed to this study as well as their structures of affiliation: Parc Zoologique et Forestier de Nouméa (PZF), the Pacific Community (SPC), Vale Inco Nouvelle-Calédonie, Direction de l'Environnement Province Sud (DENV). This study was supported by an ENS de Lyon grant for the training course of Master Students and received additional funding from the Northern Province of New Caledonia (convention n°17C376) and "CNRT Nickel et son Environnement" (CSF N°1PS2015-CNRT.IRD). The study was designed by T.B., J.N., K.B. & E.V.; Dissections were performed by J.N, C.L & K.V.; manuscript was written by T.B, J.N. & E.V.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.