Coradin

H Bouyarmane

I El Hanbali

M El Karbane

A Rami

A Saoiabi

S Saoiabi

S Masse

T Coradin

A Laghzizil
email: laghzizi@fsr.ac.ma

Parameters influencing ciprofloxacin, ofloxacin, amoxicillin and sulfamethoxazole retention by natural and converted calcium phosphates

Keywords: Antibiotics, Phosphates, Retention, Soils Capsule

Parameters influencing ciprofloxacin, ofloxacin, amoxicillin and sulfamethoxazole retention by natural and converted calcium phosphates

H Bouyarmane, I. El Hanbali, M.

Introduction

Antibiotics have been used in large quantities for several decades as human and veterinary medicine as well as husbandry growth promoters [START_REF] Moulin | A comparison of antimicrobial usage in human and veterinary medicine in France from 1999 to 2005[END_REF]. A part of administered antibiotics is excreted as the parent compound or metabolites via urine and faeces within domestic and hospital wastes [START_REF] Lin | Pharmaceutical contamination in residential, industrial and agricultural waste streams: risk to aqueous environments in Taiwan[END_REF]. They are eventually released into soils, sediments and aquatic environments through different pathways and were detected in municipal wastewater, surface water and ground water (Diaz-Cruz, 2003;[START_REF] Kumar | Antibiotic use in agriculture and its impact on the terrestrial environment[END_REF]Kümmerer, 2009a) Many of the antibiotic compounds are hazardous in nature and persist for long time in the environment, as they are poor substrates to indigenous microorganisms (Kümmerer, 2009b). In this context, it is important to evaluate the mechanisms by which they can be retained in soils [START_REF] Boxall | The sorption and transport of a sulphonamide antibiotic in soil systems[END_REF][START_REF] Thiele-Bruhn | Pharmaceutical antibiotic compounds in soils-a review[END_REF][START_REF] Chen | Effects of pH and ionic strength on sulfamethoxazole and ciprofloxacin transport in saturated porous media[END_REF][START_REF] Chen | Desorption kinetics of Sulfonamide and Trimethoprim antibiotics in soils assessed with diffusive gradients in thin-films[END_REF][START_REF] Wegst-Ulrich | Assessing antibiotic sorption in soil: a literature review and new case studies on sulfonamides and macrolides[END_REF], including through the study of their interactions with mineral phases [START_REF] Gao | Adsorption of sulphonamide antimicrobial agents to clay minerals[END_REF]G and Karthikeyan, 2005).

In parallel, such studies can be of particular relevance to predict the efficiency of mineral amendment for soil remediation purpose [START_REF] O'day | Mineral-based amendments for remediation[END_REF][START_REF] Mamindy-Pajany | Comparison of mineral-based amendments for ex-situ stabilization of trace elements (As, Cd, Cu, Mo, Ni, Zn) in marine dredged sediments: A pilot-scale experiment[END_REF] Especially, among most common mineral phase used in soil remediation, natural and synthetic calcium phosphates, especially hydroxyapatite derivatives, have been mainly studied for metal remediation [START_REF] Miretzky | Phosphates for Pb immobilization in soils : a review[END_REF][START_REF] Nzihou | Role of phosphate in the remediation and reuse of heavy metal polluted wastes and sites[END_REF]. However, recent reports suggest that calcium phosphates also hold potential for organic pollutant removal [START_REF] Bahdod | Adsorption of phenol from an aqueous solution by selected apatite adsorbents: Kinetic process and impact of the surface properties[END_REF][START_REF] Lin | Study of the adsorption of phenol from aqueous solution onto apatite nanopowders[END_REF][START_REF] Bouyarmane | Pyridine and phenol removal using natural and synthetic apatites as low cost sorbents: Influence of porosity and surface interactions[END_REF]). Yet, very little is known about the reactivity of the apatite surface towards complex molecules of environmental relevance.

In this context, the present study aims at evaluating the retention of four widespread antibiotics, ciprofloxacin, ofloxacin, amoxicillin and sulfamethoxazole by a natural phosphate rock. As a comparison, we used a synthetic porous hydroxyapatite obtained from the natural ore by a dissolution/reprecipitation process. The kinetics and isotherms of sorption were established and modeled in order to clarify the mechanisms of interactions between the antibiotics and the two mineral phases. The influence of pH on the retention efficiency was studied. The data were discussed in terms of the molecular characteristics (size, charge, hydrophobicity) of the antibiotics and surface properties (porosity, hydrophobicity) of the powders.

Materials and Methods

Chemicals

Ciprofloxacin (1-cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-yl-quinoline-3-carboxylic acid, CIP), Ofloxacin ((R,S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1piperazinyl)-7-oxo-7H-pyrido [1,2,3-de]-1, 4-benzoxazine-6-carboxylic acid, OFL) ,

Amoxicillin ((2S,5R,6R)-6-{[(2R)-2-amino-2-(4-hydroxyphenyl)-acetyl]amino}-3,3- dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, AMO) and Sulfamethoxazole (4-amino-N-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide, SUL)
were purchased from Aldrich (Fig. 1). Calculated octanol-water partition coefficients XlogP3 were obtained from the PubChem (NCBI) database (http://pubchem.ncbi.nlm.nih.gov). The Marvin 15.1.12 software (ChemAxon (http://www.chemaxon.com), was used for drawing, displaying and analyzing chemical structures.

Mineral phases

The natural phosphate (NP) used in this work was from the Bengurir region (Morocco) (El Asri et al., 2009a). The sample was washed and sieved to give a -400/+100 µm size fraction using ASTM Standard sieves. It consists of francolite, a carbonate-rich form of fluoroapatite together with ca. 2 wt% SiO 2 (quartz). The amount of organic matter is < 1.5 wt%. Its specific surface area is ca. 20 m 2 g -1 . The converted porous hydroxyapatite (CP) was obtained from the natural phosphate by a dissolution/reprecipitation method described elsewhere (El Asri et al., 2009b). It consists of a poorly crystalline powder of hydroxyapatite nanoparticles, ca 50 nm in diameter. The specific surface area (150 m 2 g -1 ) mainly originates from interparticle porosity in the mesoporous range (pore size 5-15 nm). Electron micrographs of NP and CP are provided in Supplementary Material.

Adsorption studies

Adsorption kinetics was followed in batch experiments conducted at 25°C by adding 200 mg of sorbent to 100 mL of an aqueous solution containing 20 mg L -1 of antibiotic.

The pH of the mixture was adjusted to 5.6 and the suspension kept under mechanical ! Ciprofloxacin (CIP)! MW = 331.3 g mol-1! pKa 1 = 6.1 ; pKa 2 = 8.7 ; XlogP3 = -1.1! Ofloxacin (OFL)! MW = 361.4 g mol-1! pKa 1 = 6.0 ; pKa 2 = 8.2 ; XlogP3 = -0.4! Amoxicilin (AMO)! MW = 365.4 g mol -1! pKa 1 = 2.4 ; pKa 2 = 7.4; pKa 3 = 9.6 ; XlogP3 = -2! Sulfomethoxazole (SUL)! MW = 253.3 g mol-1! pKa 1 = 1.6 ; pKa 2 = 5.7 ; XlogP3 = 0.9! stirring over 3 hours. Sorption isotherm studies were conducted in similar conditions using antibiotics concentrations in the 5-130 mg L -1 range. At selected time interval, the suspensions were sampled through direct filtration using 0.45 µm membrane filter. The antibiotic concentration in supernatant was monitored using a UV-visible spectrophotometer (Perking Elmer Lamda 35) working at λ = 230 nm, 244 nm, 273 nm, 295 nm for AMO, SUL, CIP and OFL, respectively. To study the effect of pH on antibiotics sorption, addition of HNO 3 or NaOH to the antibiotic solutions was performed. UV-visible spectra of antibiotics were recorded at all pH values in order to detect any variations in optical density at the selected wavelength and correct the concentration values accordingly For the detection of low levels of antibiotics and for the study of multiple antibiotics adsorption, high performance liquid chromatography (HPLC) was used. The conditions were: (i) CIP : Agilient-Zorbax C18 SB (250 x 4.6 mm) 5 µm ; column temperature : 40 °C, mobile phase : H 3 PO 4 -trimethylamine (pH 3):acetonitrile (87 :13), flow : 1.5 mL.min -1 ; injected volume : 50 µL ; (ii) OFL : GL Sciences Inc Inertsil C18 (250 x 4.6 mm) 5 µm ; column temperature : 45 °C ; mobile phase : CH 3 COONH 4 -NaClO 4 -H 3 PO 4 (pH 2.2) :acetonitrile (85 :15) ; flow 2 mL.min -1 ; injected volume : 100 µL ; (iii) AMO : Water Symmetry C18 (250 x 4.6 mm) 5 µm, mobile phase : NaH 2 PO 4 (pH 4.4) : H 3 PO 4 /methanol (98 :2), flow : 1 mL.min -1 ; injected volume : 100 µL ; (iv) SUL :

Water XTerra MS C18 (250 x 4.6 mm) 5 µm; column temperature : 30 °C ; mobile phase : KH 2 PO 4 (pH 5.3) : KOH/methanol (65 :35), flow : 1 mL.min -1 ; injected volume : 50 µL The amount of sorbed antibiotic was calculated by using Eq. ( 1)

q t = C 0 -C e m V (1)
where q t is the amount of adsorbed antibiotic at time t (mg g -1 ), C 0 and C(t) are the antibiotic concentration in solution at t = 0 and t = t (mg L -1 ), V is the volume (L) of the antibiotic solution and m is the weight (g) of the sorbent. All experiments were carried out in triplicate. The mean values are reported and the error range was always < 5 %.

In order to determine the kinetics parameters of the sorption reactions, Lagergren pseudo-first [START_REF] Srihari | The kinetics and thermodynamic studies of phenol-sorption on three agro-based carbons[END_REF] and pseudo-second order [START_REF] Ho | Review of second-order models for adsorption systems[END_REF] models have been applied to experimental data. The first model allows the determination of q e,1 the calculated amount of adsorbed antibiotic at equilibrium (mg g -1 ) and k 1 the first order kinetic constant (min -1 ). The second model provides q e,2 the calculated amount of adsorbed antibiotic at equilibrium (mg g -1 ) and k 2 the second order kinetic constant (g mg -1 min -1 ) Sorption isotherms were analyzed using the Langmuir and Freundlich models [START_REF] Mittal | Freundlich and Langmuir adsorption isotherms and kinetics for the removal of Tatrazine from aqueous solutions using hen feathers[END_REF]. The first model allows the calculation of q e,max the maximum amount of adsorbed antibiotic at equilibrium (mg g -1 ), and β is the Langmuir constant (L.mg -1 ) related to the adsorption energy. The second model leads to the determination of K f the Freundlich constant correlated to the maximum adsorption capacity and n the adsorption intensity, respectively.

Analyses of the calcium content of the solutions before and after antibiotic sorption was performed using inductively coupled plasma (ICP) emission spectroscopy (ICPS-7500, Shimadzu, Japan)

Results and discussion

Kinetics study of the sorption process was first conducted at low antibiotic concentration (20 mg g -1 ). SUL, CIP and OFL show a similar, fast rate of sorption over the first 30 min of contact before an equilibrium is reached (Fig. 2). In contrast, the immobilization of AMO is significantly slower, with equilibrium being observed after ca. 3h.

Fig.2

Evolution of the amount of sorbed antibiotic (q t ) with contact time t on the natural phosphate (NP) and converted (CP) phosphate powder The Langergren's pseudo second-order kinetic model (k 2 and q e,2 ) was found welladapted to reproduce the kinetics data for the first three antibiotics on both NP and CP (Table 1). The kinetics constants vary in the order k 2 (CIP) > k 2 (OFL) ≈ k 2 (SUL) for NP and k 2 (CIP) ≈ k 2 (SUL) > k 2 (OFL) for CP. In contrast, the pseudo first-order model was found more adapted to describe the AMO sorption kinetics. However, lower reliability of the fitting procedure was observed for CP data (R 2 = 0.981) compared NP (R 2 = 0.989). The retention capacity of natural and converted phosphate powders was then studied by performing sorption isotherms for initial antibiotic concentrations ranging from 5 mg L -1 to 130 mg L -1 over 3 h. In all cases, sorbent saturation was reached and experimental maximum capacities q max were found between 19 mg g -1 and 24 mg g -1 (Fig. 3). On both sorbents, the highest capacities were found for OFL molecules. Variations between the NP and CP were within the error range (5 %, i.e. ± 1 mg g -1 ) of the measurements.

Natural phosphate

Experimental q max (mg g -1 ) 21 24 22 21

Langmuir q e , max (mg g -1 ) Understanding these results requires to examine and compare the chemical properties of the four antibiotics along three parameters that can influence their sorption behaviours, i.e. dimensions, charge and hydrophilicity/hydrophobicity (Fig. 1). The first parameter can be assessed considering the molecular weight (MW) of the molecules. In this context, CIP, OFL and AMO have close MWs whereas SUL is significantly smaller.

β (L mg -1 )
Noticeably, when calculating sorption capacities per mole of antibiotic, one obtain for NP sorption isotherms, q e,max = 0.08 mmol g -1 for SUL and q e,max = 0.06 mmol g -1 for other antibiotics. This is in agreement with the fact that a given sorbent surface can, in principle, accommodate more small molecules than large ones.

Coming to the molecular charge, the pK a values of the various ionizable groups allow the determination of the main forms of each antibiotic at pH 5.6 (Fig. 4). For CIP, the amine group of the piperazinyl ring is positively charged whereas the carboxyl group is mainly in its protonated form so that this antibiotic is mainly in a cationic form.

OFL has close pK a values and is therefore also cationic. In contrast, the acidity of the COOH group of AMO is much higher so that it is deprotonated at pH 5.6. Therefore, the prevalent form of AMO is zwitterionic. Finally, SUL has two rather acidic amine groups with pK a of 1.6 and 5.7. Therefore, at pH 5.6, neutral and anionic forms are coexisting in similar amounts. In parallel, the point of zero charge of hydroxyapatite is in the pH 7-8 range [START_REF] Bell | The point of zero charge of hydroxyapatite and fluorapatite in aqueous solutions[END_REF], mainly resulting from the balance between POH/PO -and Ca 2+ groups. At pH 5.6, the surface is therefore slightly positive.

Therefore one should expect a favored adsorption of SUL compared to the three other antibiotics. In contrast, OFL and CIP should be less efficiently retained than AMO. In principle, the strength of interaction between the antibiotics and the surface can be estimated from β Langmuir parameter and 1/n Freundlich parameter. Unfortunately, the SUL sorption process could not be suitably fitted using the Langmuir model so that comparison with the three other antibiotics is difficult. Nevertheless, it must be noted that calculated 1/n values are relatively large, indicating a strong interaction between SUL and the sorbent phase. As far as the other antibiotics are concerned, one should notice that OFL sorption is systematically associated with a larger β value than CIP and AMO. Therefore another parameter must be taken into account.

Fig. 4 Calculated average charge per molecule for the four antibiotics

The XlogP3 values provide an indication of the hydrophilic/hydrophobic balance of the molecules [START_REF] Cheng | Computation of octanol-water partition coefficients by guiding and additive model with knowledge[END_REF]. On this basis, the hydrophilic character of the considered antibiotics follows the order AMO > CIP > OFL > SUL. However partition coefficients are calculated using the unionized form of the molecules, being therefore fully relevant for AMO, partially adapted for SUL and must be taken with care for CIP and OFL. A better insight can be obtained by examining the 3D molecular structure of these different antibiotics (Fig. 5). In the SUL structure, the planes of two aromatic rings form a ca. 130° angle. The terminal primary amine is easily accessible whereas the central secondary amine is buried between the aromatic rings. This explains the In this context, it is interesting to note that AMO is the only molecule whose sorption kinetics was suitably fitted by a pseudo-first order model. This means that AMO molecules are adsorbed individually on the surface, in agreement with its hydrophilic character. In contrast, aromatic hydrophobic molecules have a tendency to interact one with another in water, so that they would tend to get adsorbed as dimers (or larger aggregates). This explains why higher-order models are required to fit the kinetics data of the three other antibiotics. In parallel, the sorption isotherms of AMO, CIP and OFL are well-fitted by a Langmuir equation corresponding to a single layer deposition. This can be explained considering that for these three molecules, multi-layer stacking via intermolecular interactions of aromatic rings are sterically limited due to the presence of non-aromatic cycles pointing out of their plane. In contrast, for SUL, possible molecular stacking via π-π interactions are possible, that would explain the better suitability of the Freundlich model, that applies to heterogeneous, sometimes multi-layered sorption, to fit the experimental data.

The comparison between the two sorbents reveals that the conversion process does not induce significant modification of the maximum capacities. This can be surprising considering that the specific surface area of the powder after conversion increases from 20 m 2 g -1 to 150 m 2 g -1 . A similar observation was reported for pyridine adsorption on similar materials [START_REF] Bouyarmane | Pyridine and phenol removal using natural and synthetic apatites as low cost sorbents: Influence of porosity and surface interactions[END_REF]. It was attributed to the fact that adsorption of this molecule destabilizes the surface-coated CP nanoparticles, leading to their aggregation and therefore loss of available surface for further sorption. Another explanation lies in the hydrophobic character of francolite compared to hydroxyapatite [START_REF] Lu | Wetting of francolite and quartz and its significance in the flotation of phosphate rock[END_REF] that should favor the surface interactions of NP with the antibiotics compared to CP.

To clarify the process of sorption, the effect of pH on antibiotics sorption was studied in the 4-10 range (Fig. 6) and analyzed using the calculated pH-charge curves (Fig. 4).

As a common trend, the sorption capacity slightly increases with increasing pH for all antibiotics and for the two sorbents. For NP, these values increased of ca. 15-20 % for CIP and OFL and 20-25 % for SUL and AMO. For CP, similar values were obtained for CIP and OFL whereas sorption capacity increased by 25-30 % for SUL and AMO. As mentioned earlier, the isoelectric point of hydroxyapatite is ca. 7-8 , resulting from the contribution of Ca 2+ , OH -, and phosphates groups. CIP and OFL are mainly in a positively charged form at pH 4. With increasing pH, deprotonation of the carboxylic acid function first occurs leading to zwitterionic species until the piperazinyl rings become neutral, resulting in a major negative form above 9. SUL is mainly neutral at pH 4 and the negative form then become prevalent at higher pH due to deprotonation on the central amine group. Accordingly AMO is zwitterionic at pH 4 due to the coexistence of carboxylates and ammonium groups and the negative form becomes more abundant with increasing pH as the latter groups become neutral. At higher pH, the deprotonated phenol function further increase the negative charge of the molecule.

Thus from an general electrostatic point of view, an increase in antibiotic sorption between pH 4 and pH 7-8 can be understood for SUL and AMO considering that their negative form is becoming more abundant while hydroxyapatite remains positivelycharged, allowing for electrostatic attractive interactions to occur. For CIP and OFL, it can be suggested that the decreasing abundance of positively-charged molecular forms with increasing pH parallels the decreasing positive charge of the hydroxyapatite surface, therefore lowering electrostatic repulsive interactions. Note that attractive interactions between positively-charged molecules and deprotonated phosphates are also possible. Nevertheless, the more limited increase in sorption capacity observed for CIP and OFL compared to AMO and SUL suggest that the latter interactions are less effective than the former. Unfortunately, the low amount of sorbed antibiotics did not allow for further analysis of the sorption process by Infra-red spectroscopy (see Supplementary Material). showed a similar increase in sorption capacity above the point of zero-charge of the sorbing phase [START_REF] Bouyarmane | Pyridine and phenol removal using natural and synthetic apatites as low cost sorbents: Influence of porosity and surface interactions[END_REF]. At this time, it was suggested that neutral amines could interact with Ca 2+ ions present on the surface. As a matter of fact, all four antibiotics bear amine groups that can be involved in such attractive interactions.

Importantly, the Ca 2+ content of the supernatant of the powders was determined before and after antibiotic sorption. In all cases, this amount was lower than 0.1 mg.mL -1 , a value that must be compared with the initial 2 g.mL -1 calcium phosphate (i.e. ca. 1 g.mL -1 Ca) content. This allows to exclude any significant cation exchange reaction.

It is also worth mentioning that for SUL and AMO, the progressive formation of more negative species with increase in pH increase also correlate with a decrease in their hydrophobic character, which can favor their adsorption. This can explain why these antibiotics show a higher increase in sorption in basic media on the hydrophilic CP compared to the more hydrophobic NP.

In a step further, the adsorption of a mixture of the four antibiotics at a similar concentration (20 mg.L -1 ) was studied. The amount of sorbed molecules at equilibrium q e are shown in Fig. 7 and compared with values obtained for antibiotics alone at the same concentration. Each antibiotic was adsorbed at a 2.2-3.2 mg.g -1 content so that the total amount of adsorbed antibiotics from the mixture was ca. 10 mg.g-1. As a comparison, these value varied between 4.5 mg.g -1 and 9 mg.g -1 for individual molecules at an initial 20 mg.mL -1 concentration and reached ca. 20 mg.g -1 at an initial 80 mg.mL -1 content. Therefore, the overall sorption ability of the powders was halved when antibiotic mixtures were present. Such a decrease could be expected considering that the sorption process appears to be strongly dependent on intermolecular interactions that can be perturbed by the presence of other components. In this context, it is interesting to notice that whereas the amount of sorbed CIP was larger than that of OFL in simple solutions, this trend is reversed in mixtures, suggesting that despite their close structural and chemical features, some subtle variations in conformation and/or functional group accessibility can significantly impact on their behavior.

Fig. 7

Variation of sorbed antibiotics (q e ) as single components or from mixtures on natural (NP) and converted phosphate (CP) powders (antibiotic concentration : 20 mg.mL -1 each)

Finally, it is important to compare the sorption properties of these materials with those reported in the literature [START_REF] Homem | Degradation and removal methods of antibiotics from aqueous matrices -a review[END_REF]. For instance, for SUL, K f values (in L kg -1 ) are ca. 1-2 for sediments [START_REF] Hou | Sulfamethoxazole sorption by sediment fractions in comparison to pyrene and bisphenol A[END_REF] and 10 for clays [START_REF] Gao | Adsorption of sulphonamide antimicrobial agents to clay minerals[END_REF], therefore much higher than here-reported values. Our data suggest that this poor affinity is due to the low surface charge of francolite and hydroxyapatite in the 5-7 pH range, preventing strong attractive electrostatic interactions with here-studied antibiotics. In these conditions, it is expected that calcium phosphates, being natural or synthetic, do not significantly contribute to the immobilization of here-studied However, we evidenced the importance of the hydrophobic character of these molecules on their sorption, an observation that can apply to a large range of bioactive agents exhibiting lipophilic properties. These data can contribute to a better understanding of the fate of antibiotics in soils, a field where the influence of the hydrophobic character of the drug is so far poorly understood. In this context, francolite rocks appear as particularly interesting material for further studies, as a major form of natural calcium phosphates.

Fig. 1

 1 Fig.1 Chemical formula, molecular weights (MW), relevant pKa values and calculated

Fig. 3

 3 Fig.3Variation of sorbed antibiotic content ( q e ) with antibiotic concentration at

Fig. 5

 5 Fig. 5 Molecular structure of the antibiotics and their proposed mode of adsorption on

Fig. 6

 6 Fig.6Variation of sorbed antibiotics (q e ) with pH on the natural (NP) and converted

  antibiotics in soils, in agreement with the literature[START_REF] Wegst-Ulrich | Assessing antibiotic sorption in soil: a literature review and new case studies on sulfonamides and macrolides[END_REF].

Table 1

 1 Kinetic rate constants (k) and adsorption capacities (q) as obtained for different models for antibiotic sorption by the natural and converted phosphate powder. R 2 indicate the correlation coefficients for the linear fits.

	Kinetic model Parameter	CIP	OFL	AMO	SUL
		Natural phosphate

Table 2

 2 Table2). Calculated q e,max values were in good agreement with experimental values. The largest β value was obtained for OFL while this parameter had a similar value for CIP and AMO. In the case of SUL, the Freundlich model was found more adapted to fit the experimental data. Noticeably, a systematic decrease in β and R 2 values for OFL, CIP and AMO was obtained for CP compared to NP. The same trend was evidenced for K f and R 2 values calculated from modeling of SUL sorption data, while 1/n increased. Langmuir and Freundlich constants for antibiotics sorption on the natural

	and converted phosphate powder
	CIP	OFL	AMO	SUL

Acknowledgments

The authors thank the CNRS for funding this research through the APATENV project of the PICS program. T.C and S. M. are very thankful to M. Selmane and F. Tielens (LCMCP) for their assistance in the determination of molecular structures.

Appendix A. Supplementary Material

Electron microscopy images of the natural and converted phosphate powders. Selected FTIR spectra of the powders after antibiotic sorption.


Atsushi Matsuoka
email: atsushi.matsuoka@takuvik.ulaval.ca

Eva Ortega-Retuerta
email: ortegaretuerta@icm.csic.es

Annick Bricaud
email: annick@obs-vlfr.fr

Kevin R Arrigo
email: arrigo@stanford.edu

Marcel Babin
email: marcel.babin@takuvik.ulaval.ca

Characteristics of colored dissolved organic matter (CDOM) in the Western Arctic Ocean: relationships with microbial activities

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

(WAO) from early to late summer as part of the MALINA and the ICESCAPE expeditions. The data set covered contrasting situations, from bloom to post-bloom conditions and from riverinfluenced to oceanic water masses. While CDOM photobleaching occurred in the surface layer (< 20 m), we observed significantly lower spectral slopes for CDOM absorption spectra (S CDOM ) in addition to higher a CDOM (440) in the layer below (intermediate layer: 30.7 < salinity < 33.9). In particular, the low S CDOM values were found in the Chukchi Sea and the western part of the Beaufort Sea, which coincided with high BP and BA values. Considering the high primary production observed in these areas during our cruises [START_REF] Arrigo | Massive Phytoplankton Blooms Under Arctic Sea Ice[END_REF], we hypothesize that S CDOM variations reflect the degradation of phytoplankton that is associated with heterotrophic bacterial activity. In our datasets, a simple regression analysis showed that S CDOM was significantly correlated with BP and BA. A principal component analysis further supported this conclusion. From our field observations, it was shown that variations in a CDOM (440) and S CDOM result to a large extent from bacterial activity, at least in the WAO.

Introduction

Examining the budget of dissolved organic carbon (DOC) in the Arctic Ocean is crucial to improving our understanding of modifications in the carbon cycle resulting from ongoing global warming. While this warming likely induces thawing of permafrost containing a huge amount of DOC, which is subsequently delivered by river discharge into the Arctic Ocean [START_REF] Peterson | Increasing river discharge to the Arctic Ocean[END_REF][START_REF] Mcclelland | A pan-arctic evaluation of changes in river discharge during the latter half of the 20 th century[END_REF][START_REF] Raymond | Flux and age of dissolved organic carbon exported to the Arctic Ocean: A carbon isotopic study of the five largest arctic rivers[END_REF], the long-term trend in the DOC budget of the Pan-Arctic Ocean has yet to be established. In recent studies, [START_REF] Matsuoka | Estimating absorption coefficients of colored dissolved organic matter (CDOM) using a semi-analytical algorithm for southern Beaufort Sea waters: applications to deriving concentrations of dissolved organic carbon from space[END_REF][START_REF] Matsuoka | A systhesis of light absorption properties of the Arctic Ocean: application to semianalytical estimates of dissolved organic carbon concentrations from space[END_REF] developed a semi-analytical algorithm to quantitatively estimate DOC concentrations for Arctic coastal waters using satellite remote sensing data, which allows the continuous monitoring of variability in DOC concentrations. In contrast, knowledge about the production and the removal processes gained from field observations using traditional methods is limited temporally and geographically [START_REF] Bussmann | Bacterial utilization of humic substances from the Arctic Ocean[END_REF][START_REF] Garneau | Seasonal dynamics of bacterial biomass and production in a coastal arctic ecosystem: Franklin Bay, western Canadian Arctic[END_REF][START_REF] Kirchman | Microbial growth in the polar oceansrole od temperature and potential impact of climate change[END_REF]Ortega-Retuerta et al., 2012). This prevents us from understanding the balance between these processes.

Light absorption by the colored fraction of dissolved organic matter (CDOM) provides useful information about biogeochemical processes [START_REF] Carder | Marine humic and fulvic acids: Their effects on remote sensing of ocean chlorophyll[END_REF][START_REF] Nelson | Seasonal dynamics of colored dissolved material in the Sargasso Sea[END_REF][START_REF] Miller | Determination of apparent quantum yield spectra for the formation of biologically labile photoproducts[END_REF][START_REF] Nelson | Production of chromophoric dissolved organic matter by Sargasso Sea microbes[END_REF][START_REF] Nelson | Hydrography of chromophoric dissolved organic matter in the North Atlantic[END_REF][START_REF] Matsuoka | Tracing the transport of colored dissolved organic matter in water masses of the Southern Beaufort Sea: relationship with hydrographic characteristics[END_REF]. While microbial activity is highly variable in natural environments (e.g., [START_REF] Azam | The Ecological Role of Water-Column Microbes in the Sea[END_REF], the link between heterotrophic bacterial production and CDOM absorption was reported in the Sargasso Sea [START_REF] Nelson | Seasonal dynamics of colored dissolved material in the Sargasso Sea[END_REF]. More recently, [START_REF] Matsuoka | Tracing the transport of colored dissolved organic matter in water masses of the Southern Beaufort Sea: relationship with hydrographic characteristics[END_REF] suggested that lower spectral slopes of CDOM absorption spectra observed in some Arctic Ocean water masses resulted from heterotrophic microbial activity.

However, a direct relationship between CDOM and bacteria has not been well documented.

The objective of this study is to examine the relationships between CDOM absorption properties and heterotrophic bacterial production (BP) and abundance (BA) in the Western Arctic Ocean (WAO) as well as their link with environmental variables (i.e., temperature, salinity, and nitrate and chlorophyll a concentrations). [START_REF] Ortega-Retuerta | Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance. Production and respiration on the Beaufort Sea margin[END_REF], waters in the Chukchi-Beaufort Seas during the ICESCAPE cruises were highly productive [START_REF] Arrigo | Massive Phytoplankton Blooms Under Arctic Sea Ice[END_REF]. To examine the general characteristics in the WAO, these three datasets were combined and used in this study.

Datasets and methods

Data

Temperature and salinity profiles were obtained using a SBE-911 plus (Seabird) conductivitytemperature-depth (CTD) probe. Chlorophyll a (chl a) and phaeopigment concentrations (mg m -3 ) were determined fluorometrically [START_REF] Holm-Hansen | Fluorometric Determination of Chlorophyll[END_REF]. Nitrate concentrations (NO 3 , µmol kg -1 ) were measured following Grasshoff et al. (1999) for MALINA and Armstrong et al. (1967) for ICESCAPE cruises. Oxygen concentrations (Oxy, µmol kg -1 ) were measured following [START_REF] Carpenter | The accuracy of the Winkler method for dissolved oxygen[END_REF] with modifications by [START_REF] Culberson | Dissolved Oxygen, WHP Operations and Methods[END_REF].

Apparent oxygen utilization (AOU, µmol kg -1 ) was calculated by referring solubility of oxygen for well-mixed winter waters having temperature and salinity of -1.8 °C and 31, respectively. These reference data were proven to be valid for western Arctic waters [START_REF] Matsuoka | Tracing the transport of colored dissolved organic matter in water masses of the Southern Beaufort Sea: relationship with hydrographic characteristics[END_REF].

CDOM absorption

Details of the method used for measurement of CDOM absorption are documented in [START_REF] Matsuoka | Tracing the transport of colored dissolved organic matter in water masses of the Southern Beaufort Sea: relationship with hydrographic characteristics[END_REF]. Briefly, water samples were filtered using 0.2 µm pore-size filters immediately after sampling. Absorption coefficients of CDOM (a CDOM (λ), m -1 ) were determined from 200 to 735 nm in 1-nm increments using a liquid waveguide system (UltraPath, World Precision Instruments, Inc.).

The spectral slope of a CDOM (λ) (S CDOM , nm -1 ) was calculated by nonlinear regression of the data from 350 to 500 nm [START_REF] Babin | Variations in the light absorption coefficients of phytoplankton, nonalgal particles, and dissolved organic matter in coastal waters around Europe[END_REF][START_REF] Matsuoka | Seasonal variability in the light absorption properties of western Arctic waters: parameterization of the individual components of absorption for ocean color applications[END_REF][START_REF] Matsuoka | Tracing the transport of colored dissolved organic matter in water masses of the Southern Beaufort Sea: relationship with hydrographic characteristics[END_REF].

A previous study [START_REF] Helms | Absorption spectral slopes and slope ratios as indicators of molecular weight, sources, and photobleaching of chromophoric dissolved organic matter[END_REF] showed that the spectral slopes (S 275-295 and S 350-400 , nm -1 for both) corresponding to two distinct wavelength ranges (i.e., 275-295 nm and 350-400 nm, respectively) and their ratio (S R , dimensionless) provide insights into sources (e.g., marine or terrestrial sources; [START_REF] Carder | Marine humic and fulvic acids: Their effects on remote sensing of ocean chlorophyll[END_REF][START_REF] Nelson | Hydrography of chromophoric dissolved organic matter in the North Atlantic[END_REF] and/or local processes affecting the CDOM distribution (e.g., lateral transport, vertical mixing, photo-bleaching, heterotrophic bacterial alteration; [START_REF] Nelson | Seasonal dynamics of colored dissolved material in the Sargasso Sea[END_REF][START_REF] Nelson | Production of chromophoric dissolved organic matter by Sargasso Sea microbes[END_REF][START_REF] Matsuoka | Tracing the transport of colored dissolved organic matter in water masses of the Southern Beaufort Sea: relationship with hydrographic characteristics[END_REF][START_REF] Yamashita | Photobleaching as a factor controlling spectral characteristics of chromophoric dissolved organic matter in open ocean[END_REF]. These slope parameters were calculated for our Arctic datasets by fitting a linear model to CDOM absorption coefficients in the two distinct spectral ranges [START_REF] Helms | Absorption spectral slopes and slope ratios as indicators of molecular weight, sources, and photobleaching of chromophoric dissolved organic matter[END_REF].

Bacterial abundance

Heterotrophic prokaryotes, including bacteria and archaea, are abbreviated throughout the manuscript as "bacteria". Samples were fixed with glutaraldehyde (0.25% final concentration) and stored at -80ºC until processing. During MALINA, bacterial cells (BA) were counted aboard the ship by flow cytometry using a FACS ARIA (Becton, Dickinson and company) equipped with 488 nm and 633 nm lasers and a standard filter setup [START_REF] Ortega-Retuerta | Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance. Production and respiration on the Beaufort Sea margin[END_REF]. During ICESCAPE 2010, bacterial cells were counted by flow cytometry at the home laboratory using a BD FACS Calibur Flow Cytometer (Becton, Dickinson and company). During ICESCAPE 2011, bacterial cells were counted aboard the ship using an Accuri C6 (Becton, Dickinson and company) equipped with a 488 nm laser. In all cases, samples were thawed and SYBR Green-I was added at a final dilution of 1:10,000. Samples were incubated in the dark for 15 min before analysis. Bacterial cells were identified on a plot of green fluorescence (515-545 nm) versus right angle light scatter (SSC), using the green fluorescence as a threshold parameter. High nucleic acid (HNA) and low nucleic acid (LNA) bacteria were discriminated according to their green fluorescence and counted separately [START_REF] Marie | Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I[END_REF]. HNA cells have often been considered as active bacteria [START_REF] Gasol | Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria[END_REF].

Bacterial production

Bacterial production (BP) was measured following [START_REF] Ortega-Retuerta | Carbon fluxes in the Canadian Arctic: patterns and drivers of bacterial abundance. Production and respiration on the Beaufort Sea margin[END_REF]. Briefly BP was measured by 3 H-leucine incorporation [START_REF] Smith | A simple, economical method for measuring bacterial protein synthesis rates in seawater using 3H-leucine[END_REF]. Samples (1.5 mL in triplicate plus one killed control) were added to sterile microcentrifuge tubes, containing 20-30 nM [4,5-3 H]leucine. This concentration was sufficient to saturate bacterial leucine uptake (data not shown).

Incorporation rates were measured after 2-h incubations at in situ temperature, and incubations were stopped by the addition of trichloroacetic acid (5% final concentration). Leucine incorporation rates were converted to carbon production using the conversion factor of 1.5 kg C produced per mole of leucine incorporated [START_REF] Kirchman | Leucine incorporation as a measure of biomass production by heterotrophic bacteria[END_REF], considering no isotope dilution.

Statistical analyses

Regression analysis

To examine a direct relationship between two variables, several regression analyses were performed. Because the two variables were random (or not controlled), Model II regression was applied in this study [START_REF] Legendre | Numerical Ecology[END_REF].

Principal component analysis

For a dataset containing several variables, a multiple regression analysis is not always the best method for examining the relationships among the variables. Principal component analysis (PCA) is preferable because it summarizes, in a few dimensions, most of the variability present in a dispersion matrix of a large number of variables [START_REF] Legendre | Numerical Ecology[END_REF], and has been applied to a large number of oceanographic studies (e.g., [START_REF] Legendre | Numerical Ecology[END_REF][START_REF] Uitz | Relating phytoplankton photophysiological properties to community structure on large scales[END_REF][START_REF] Suzuki | Year-round accumulation of particulate organic matter in the estuarine turbidity maximum: comparative observations in three macrotidal estuaries (Chikugo, Midori, and Kuma Rivers), southwestern Japan[END_REF]. We thus applied this method to our datasets.

Results and discussion

Vertical distribution of CDOM

To examine the vertical distribution of CDOM absorption properties in the WAO, a CDOM (440) and S CDOM were plotted against depth (Figure 3). At depth < 150 m, a CDOM (440) values were highly variable for early to middle summer (ICESCAPE2010 and 2011 data: blue crosses and red diamonds in Figure 3a andc). Values for late summer (MALINA data: black circles in Figure 3a), however, were less variable and low near the surface (down to 0.0197 m -1 ), except for river waters that showed significantly higher values (up to 1.08 m -1 ; see arrow in Figure 3a). At depth > 150 m, all data tended to decrease with depth, approaching 0.0277 ± 0.0025 m -1 (dotted rectangle in Figure 3c). This type of profile was similar to that reported by [START_REF] Guéguen | The nature of colored dissolved organic matter in the southern Canada Basin and East Siberian Sea[END_REF], who suggested that the maximal values around 150 m are associated with microbial activity (see sections 3.3 and 3.4).

While determination of the origin of CDOM (e.g., production by phytoplankton, heterotrophic bacteria, etc) is still challenging using our dataset alone, we acknowledge that phytoplankton especially at the deep chlorophyll maximum is one of the CDOM sources [START_REF] Matsuoka | Tracing the transport of colored dissolved organic matter in water masses of the Southern Beaufort Sea: relationship with hydrographic characteristics[END_REF].

Similarly to a CDOM (440), S CDOM values for early-middle summer varied widely at depths < 150 m (Figure 3b). In contrast, those values showed less variability in late summer and were highest near the surface (up to 0.022 nm -1 ; Figure 3b andd). At depths > 150 m, all S CDOM values approached 0.0167 ± 0.0005 nm -1 with increasing depth (dotted rectangle in Figure 3d).

For surface waters, our results thus showed that a CDOM (440) values decreased in association with increases in S CDOM from early to late summer (for up to 2.5 months). It is well known that as a result of solar irradiation, high-weight molecules are transported into low-weight molecules that absorb light in the shorter spectral wavelengths, a phenomenon called photo-bleaching. The spectral slope therefore increases after the photo-bleaching [START_REF] Twardowski | Photobleaching of aquatic dissolved materials: Absorption removal, spectral alteration, and their interrelationship[END_REF]. The timescale is likely one to three months [START_REF] Granskog | Coastal conduit in southwestern Hudson Bay (Canada) in summer: Rapid transit of freshwater and significant loss if colored dissolved organic matter[END_REF]. Satellite-derived sea ice concentration images further showed that surface waters at most of our sampling stations were icefree and therefore exposed to solar irradiation (Figure 2). All these results demonstrate photobleaching occurred during our observations.

For deep waters, CDOM absorption properties were stable (Figure 3a andb). Because CDOM in these waters is considered to be biologically unavailable or refractory, those absorption values can be considered as an end-member for refractory CDOM. If so, the proportion of CDOM lability could be optically quantified when the other end-member from labile CDOM is obtained. Further work is necessary to examine this issue.

Relationship between CDOM and hydrography

Because salinity can be a good proxy of Arctic Ocean hydrography (e.g., [START_REF] Carmack | Water mass structure and boundaries in the Mackenzie shelf estuary[END_REF][START_REF] Macdonald | Composition and modification of water masses in the Mackenzie shelf estuary[END_REF][START_REF] Matsuoka | Tracing the transport of colored dissolved organic matter in water masses of the Southern Beaufort Sea: relationship with hydrographic characteristics[END_REF], relationships between CDOM absorption properties (i.e., a CDOM (440) and S CDOM ) and salinity were examined (Figure 4). At salinity < 28, two types of waters were distinguished: 1) river waters with a strong negative correlation between a CDOM (440) and salinity and 2) ice-melt waters showing both low a CDOM (440) and salinity (Figure 4a; see also [START_REF] Matsuoka | Tracing the transport of colored dissolved organic matter in water masses of the Southern Beaufort Sea: relationship with hydrographic characteristics[END_REF]. These waters were observed in the Mackenzie shelf-basin area in the late summer (black circles: MALINA cruise); river waters samples were collected in the Mackenzie River mouth, while ice melt waters samples were taken near the sea ice, far away from the river mouth (> 300 km). Data points at salinities < 28 located in the Chukchi-Beaufort Seas in mid-summer (red diamonds: ICESCAPE 2011 cruise) were not far away from the negative a CDOM (440) versus salinity relationship for MALINA cruise (Figures 4a andb). In this salinity range, S CDOM exhibited low variability (0.0196 ± 0.0011; Figure 4b), which is consistent with the value reported by [START_REF] Matsuoka | Tracing the transport of colored dissolved organic matter in water masses of the Southern Beaufort Sea: relationship with hydrographic characteristics[END_REF] (0.0192 ± 0.0011).

At salinities > 28, a CDOM (440) values for the MALINA cruise tended to be lower than those for the ICESCAPE 2010&2011 cruises (early-middle summer) conducted in the Chukchi-Beaufort Seas (Figure 4c). This was especially true in the surface layer (i.e., 28 < salinity < 30.7), where the lower a CDOM (440) values for the MALINA cruise corresponded to slightly but significantly higher S CDOM values (0.0189 ± 0.0006) compared to those for the ICESCAPE 2010&2011 cruises (0.0182 ± 0.0004 and 0.0183 ± 0.0012, respectively) (T-test, p < 0.0001; Figure 4c andd), which suggests that photo-bleaching occurred from early-middle to late summer [START_REF] Nelson | Seasonal dynamics of colored dissolved material in the Sargasso Sea[END_REF][START_REF] Twardowski | Photobleaching of aquatic dissolved materials: Absorption removal, spectral alteration, and their interrelationship[END_REF]Donaghay, 2002, Matsuoka et al., 2011; see also Figure 3). In other words, CDOM in the Chukchi-Beaufort Seas observed during the early-middle summer cruises might have been relatively new because of in situ production and/or input from the Bering Sea [START_REF] Matsuoka | Seasonal variability in the light absorption properties of western Arctic waters: parameterization of the individual components of absorption for ocean color applications[END_REF][START_REF] Shen | Dissolved organic matter composition and bioavailability reflect ecosystem productivity in the Western Arctic Ocean[END_REF].

The negative relationship between a CDOM (440) and salinity in the Lower Halocline Water (LHW:

33.9 < salinity < 34.7) and Atlantic Layer (AL: salinity > 34.7; our water samples were always collected at depths shallower than 850 m) was very similar among cruises (Figure 4c). This relationship was likely stable across ice-free seasons and areas of the WAO. So the a CDOM (440)

versus salinity relationship could be specific to these waters.

In the intermediate layer between the surface and the LHW+AL layers (i.e., 30.7 < salinity < Note, however, that this result is contrary to the one obtained by [START_REF] Nelson | Production of chromophoric dissolved organic matter by Sargasso Sea microbes[END_REF]. This contrast might originate from differences in substrate of organic matter utilized by bacteria and/or in distinct bacterial assemblages. Considering these findings, we hypothesized that the significantly low S CDOM values observed in this study are associated with heterotrophic microbial activity.

To test this hypothesis, a vertical section of CDOM absorption properties, as well as salinity and phaeopigments in the intermediate layer (i.e., 30.7 < salinity < 33.9), along the transect from the Kotzebue Sound (KS) to the Chukchi Hotspot (CH) was further analyzed (Figure 5).

Relationship between the spectral slope of CDOM absorption and bacteria

To examine the direct relationship between the spectral slope of CDOM absorption and bacteria, S CDOM was regressed against BP and BA (Figure 6; chl a concentrations, temperature and nitrate concentrations were also regressed against S CDOM ). There was a weak but significant negative correlation between BP and S CDOM (Figure 6a; r 2 = 0.20, p < 0.0001). The coefficient of determination for this relationship was the second highest following the BP versus chl a relationship (r 2 = 0.24, p < 0.0001; Table 1). Temperature showed the third highest correlation with slightly lower coefficient of determination (r 2 = 0.18, p < 0.0001). Significant correlations were not found for BP versus salinity or nitrate concentrations.

S CDOM was negatively correlated with BA (Figure 6b; r 2 = 0.26, p < 0.0001). The coefficient of determination for this relationship was the highest, followed by BA versus chl a (r 2 = 0.22, p < 0.0001; Table 1). Similarly to BP, temperature showed the third highest correlation with BA (r 2 = 0.17, p < 0.0001). No significant correlations were found between BA and either salinity or NO 3 concentration. These results suggest that low S CDOM values were generally associated with high BP and BA in our environments.

A similar regression analysis was performed for the spectral parameters S 275-295 , S 350-400 , and S R , as done by [START_REF] Helms | Absorption spectral slopes and slope ratios as indicators of molecular weight, sources, and photobleaching of chromophoric dissolved organic matter[END_REF]. Although results using S 350-400 were similar to those using S CDOM (Table 2), none of them revealed higher correlations with BP and BA than S CDOM ; S CDOM is a better variable to reflect bacterial activity.

Multiple relationships among variables

To examine relationships among several variables, a PCA analysis was performed. The extracted axes of the PCA (Figure 7) can be interpreted as follows. Principal Component 1 (PC1) made the largest contribution, explaining 30.9 % of total variance. The positive component was strongly related to chl a, a CDOM (440), BA, and BP and had a weak relationship with temperature and salinity.

Thus, the positive PC1 was considered to reflect production of organic matter. More interestingly, S CDOM alone showed the opposite trend compared to the above-mentioned variables (i.e., production of organic matter), suggesting that negative PC1 might reflect the decomposition of organic matter.

Principal Component 2 (PC2) explained 21.4 % of total variance. The negative component was strongly related to NO 3 concentration and apparent oxygen utilization (AOU) and was weakly related to salinity. NO 3 and AOU were tightly correlated, consistent with recent findings by [START_REF] Matsuoka | Tracing the transport of colored dissolved organic matter in water masses of the Southern Beaufort Sea: relationship with hydrographic characteristics[END_REF] for these two variables in the salinity range of 30.7-33.9 (or approximately 50-200 m depth; see their Figures 3c andd). Thus, negative PC2 was considered to reflect the aphotic zone. In contrast, the positive PC2 might reflect the euphotic zone, which is further supported by the fact that this component was weak but related to the production of organic matter.

Note that result of salinity in the PCA analysis is not surprising and can be explained as follows.

First, salinity exhibited an opposite trend compared to that shown by S CDOM . This general trend is shown in Figure 4. Second, this variable had a negative direction in PC2. This result is consistent with our discussion that negative PC2 represents the aphotic zone, showing higher salinity.

Furthermore, Figure 7 provides important information: Because only S CDOM showed opposite direction compared to parameters related to production of organic matter, this variable might be considered to reflect the degradation products of organic matter.

As expected, S CDOM was negatively correlated with BP and BA (Figure 6), which is consistent with the result of simple regression analysis (Figure 6 and Table 1). Similarly, S CDOM was negatively correlated with chl a and phaeopigments (p < 0.0001 for both). By taking into account the findings in Figures 567, our results suggest that variations in S CDOM reflect the degradation of phytoplankton that is associated with heterotrophic bacterial activity.

Conclusions

This study demonstrated that variations in the spectral slope pf CDOM absorption (S CDOM ) are partly explained by bacterial production (BP) and bacterial abundance (BA) variations. A simple regression analysis showed that S CDOM was related to both BA and BP, which was further supported by PCA analysis. Bacterial abundance and production is likely dependent on bioavailability of DOM (e.g., [START_REF] Moran | Carbon loss and optical property changes during long-term photochemical and biological degradation of estuarine dissolved organic matter[END_REF][START_REF] Helms | Absorption spectral slopes and slope ratios as indicators of molecular weight, sources, and photobleaching of chromophoric dissolved organic matter[END_REF][START_REF] Ortega-Retuerta | Biogeneration of chromophoric dissolved organic matter by bacteria and krill in the Southern Ocean[END_REF]. The spectral slope of CDOM reflects to some extent the level of DOC availability [START_REF] Moran | Carbon loss and optical property changes during long-term photochemical and biological degradation of estuarine dissolved organic matter[END_REF][START_REF] Helms | Absorption spectral slopes and slope ratios as indicators of molecular weight, sources, and photobleaching of chromophoric dissolved organic matter[END_REF][START_REF] Ortega-Retuerta | Biogeneration of chromophoric dissolved organic matter by bacteria and krill in the Southern Ocean[END_REF]. Therefore, it is consistent that S CDOM is significantly correlated with bacterial activity. Further work is necessary to better understand changes in CDOM cycling in the Arctic Ocean. These images were generated by averaging daily images available during each cruise.

Sampling stations are also displayed with white circles, yellow crosses, and red diamonds, respectively. x-axis) and 2 (PC2: y-axis) for a given variable and 2) magnitude of an arrow represents the strength of the variable to the components. For example, BP and chl a concentrations showed a similar direction, suggesting they are related to each other and to positive PC1.

This result is consistent with the direct regression analysis (Figure 6 and Table 1). The positive PC1 reflects production of organic matter (section 3.4). Thus, BP and chl a concentrations can be considered as contributors to the production of organic matter. See section 3.4 for details. Table 1. Summary of the Model II regression analysis for bacterial production (BP, µgC m -3 d -1 ) and abundance (BA, cells ml -1 ) as a function of chlorophyll a (chl a, mg m -3 ) concentrations, spectral slope of CDOM (S CDOM , nm -1 ), temperature (T, degrees C), salinity, and nitrate (NO 3 , µmol kg -1 ) concentrations. A total of 133 samples were used for each regression analysis.

Parameter

  were collected during three cruises in the WAO (Figure 1): the joint France-Canada-USA Arctic campaign, MALINA (30 July to 27 August 2009), and the National Aeronautics and Space Administration (NASA) ICESCAPE cruises in 2010 and 2011 (referred to as ICESCAPE 2010: 15 June to 22 July 2010 and ICESCAPE 2011: 25 June to 29 July 2011, respectively). While waters in the Mackenzie shelf-basin area during the MALINA cruise were oligotrophic and typical of postbloom conditions

  2.5. Sea ice concentrationDaily sea ice concentrations data acquired by the Defense Meteorological Satellite Program (DMSP) SSM/I passive microwave sensor (25-km spatial resolution) were downloaded from the /sidads.colorado.edu/pub/DATASETS/nsidc0051_gsfc_nasateam_seaice/. Daily images (29 for MALINA, 38 for ICESCAPE2010, and 35 for ICESCAPE2011) were averaged to generate an image of mean sea ice concentrations for each cruise (Figure2).

  33.9), a CDOM (440) values for the ICESCAPE 2010&2011 cruise in the Chukchi-Beaufort Seas were significantly higher than those for MALINA in the Mackenzie shelf-basin area (T-test, p < 0.0001).Correspondingly, S CDOM values in the intermediate layer were much lower for the ICESCAPE 2010&2011 cruises than for the MALINA cruise (T-test, p < 0.0001; Figure4d). Based on field observations,[START_REF] Nelson | Seasonal dynamics of colored dissolved material in the Sargasso Sea[END_REF] suggested that bacteria produce CDOM when taking up dissolved organic carbon (DOC), which is also consistent with results from laboratory experiments.[START_REF] Moran | Carbon loss and optical property changes during long-term photochemical and biological degradation of estuarine dissolved organic matter[END_REF],[START_REF] Helms | Absorption spectral slopes and slope ratios as indicators of molecular weight, sources, and photobleaching of chromophoric dissolved organic matter[END_REF][START_REF] Ortega-Retuerta | Biogeneration of chromophoric dissolved organic matter by bacteria and krill in the Southern Ocean[END_REF] observed that heterotrophic bacterial activity is associated with a decrease in the spectral slope of CDOM absorption over time.

  The transect covered both river-influenced and biologically productive areas sampled during the ICESCAPE 2010 cruise (see Figure 1 for location). Waters showing high a CDOM (440) and low salinity values were observed in the surface layer of the KS, indicating river-influenced waters (Figure 5a and b).Further offshore, this trend was no longer observed in the surface layer of the CH. Relatively high a CDOM (440) values, corresponding to significantly low S CDOM values, were observed near the bottom of the CH. Interestingly, the low S CDOM values were associated with high concentrations of phaeopigments (r 2 = 0.70, p < 0.0001; Figure5c and d); BP measurements are not shown here because of the limited number of data points along this transect. Because phaeopigments reflect degradation products of phytoplankton intermediated by bacteria, this result partly supports our hypothesis that both the high a CDOM (440) and low S CDOM values observed in this study resulted from heterotrophic microbial activity.

Figure 1 .

 1 Figure 1. Locations of sampling stations for ICESCAPE 2010 (blue crosses), ICESCAPE 2011 (red

Figure 2 .

 2 Figure 2. Mean sea ice concentration images provided by the using satellite microwave sensor,

Figure 3 .

 3 Figure 3. Vertical profiles of (a) CDOM absorption coefficients at 440 nm (a CDOM (440), m -1 ) and (b)their spectral slope (S CDOM , nm -1 ). X-axis for (a) is log-transformed to show variability in

Figure 4 .

 4 Figure 4. Upper panels: CDOM absorption coefficients at 440 nm as a function of salinity (S) for

Figure 5 .

 5 Figure 5. Vertical sections of (a) salinity, CDOM absorption properties (b) a CDOM (440) and (c)

Figure 6 .

 6 Figure 6. Relationship between S CDOM and (a) bacterial production (BP, µg C m -3 d -1 ) and (b)

Figure 7 .

 7 Figure 7. Biplot of principal component analysis (PCA). The important features of this plot are as

Figure 1

 1 Figure 1

  Figure 2

  Figure 4

Figure 5

 5 Figure 5

  Figure 6

  For conversion to a base 10 logarithm, 2 was added to T. Table2. Determination coefficients between bacterial variables (BP or BA) and spectral slope parameters (S 275-295 , S 350-400 , and its ratio S R ) proposed by[START_REF] Helms | Absorption spectral slopes and slope ratios as indicators of molecular weight, sources, and photobleaching of chromophoric dissolved organic matter[END_REF].

		Slope	0.06	0.04
		p-value	< 0.05	< 0.01
	NO 3	r 2	0.04	0.05
		Slope	-0.03	-0.01
		p-value	< 0.05	< 0.01
	r 2	Log 10 (BP)	Log 10 (BA)
	S 275-295	0.11	0.12
	S 350-400	0.19	0.23
	S R	0.03	0.04
		Statistics	Log 10 (BP)	Log 10 (BA)
	Log 10 (chl a)	r 2	0.24	0.22
		Slope	0.40	0.15
		p-value	< 0.0001	< 0.0001
	S CDOM	r 2	0.20	0.26
		Slope	-169.9	-73.65
		p-value	< 0.0001	< 0.0001
	Log 10 (T+2) *	r 2	0.18	0.17
		Slope	0.70	0.26
		p-value	< 0.0001	< 0.0001
	Salinity	r 2	0.04	0.10

*

Acknowledgements

We are grateful to the captain and crews of the Canadian Icebreaker CCGS Amundsen and the US icebreaker USCGC Healy. CTD deployment and data processing were made by Y. Gratton, L. Prieur, and C. Marec for MALINA and J. Swift and R. S. Pickart for ICESCAPE cruises. Water samples from a small boat were provided by S. B. Hooker. Fluorometric chlorophyll a concentrations were measured by S. Bélanger and A. Mignot for MALINA and K. R. Arrigo, M. Mills, G. van Dijken, Z. Brown, M. Palmer, and K. Lowry for ICESCAPE cruises. Nitrate concentrations were provided by J. E. Tremblay, P. Raimbault, N. Garcia, and J. Gagnon for MALINA and S. Becker for ICESCAPE cruises. Technical supports by Q. Allison, S. Tolley, S. Hiller, S. Laney, and S. Roberts are much appreciated. Bacterial abundance and production data during MALINA were kindly provided by D. Marie, F. Joux and W.H. Jeffrey. Comments from two anonymous reviewers greatly improved the manuscript. This study was conducted as part of the MALINA Scientific Program funded by ANR (Agence nationale de la recherche), INSU-CNRS (Institut national des sciences de l'univers -Centre national de la recherche scientifique), CNES (Centre national d'études spatiales) and ESA (European Space Agency). This research was also supported by the Ocean Biology and Biogeochemistry Program and the Cryosphere Science Program of the National Aeronautic and Space Administration (NNX10AF42G to K. Arrigo). We also thank a joint contribution to the research programs of UMI Takuvik, ArcticNet (Network Centres of Excellence of Canada) and the Canada Excellence Research Chair in Remote Sensing of Canada's New Arctic Frontier. References Armstrong, F.A. Stearns, J., C. R., and Strickland, J. D. H., 1967, The measurement of upwelling