Organic Geochemistry 75 (2014) 61–73

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Organic Geochemistry

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Degradation of sterols and terrigenous organic matter in watersof the Mackenzie Shelf, Canadian Arctic

http://dx.doi.org/10.1016/j.orggeochem.2014.06.0020146-6380/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Aix-Marseille University, Mediterranean Institute ofOceanography (MIO), 13288, Marseille, Cedex 9; Université du Sud Toulon-Var,83957, CNRS-INSU/IRD UM 110, France. Tel.: +33 4 91 82 96 51; fax: +33 4 91 82 9641.

E-mail address: jean-francois.rontani@univ-amu.fr (J.-F. Rontani).

Jean-François Rontani a,⇑, Bruno Charrière a,b, Richard Sempéré a, David Doxaran c, Frédéric Vaultier a,Jorien E. Vonk d,e, John K. Volkman f

a Aix-Marseille University, Mediterranean Institute of Oceanography (MIO), 13288, Marseille, Cedex 9; Université du Sud Toulon-Var, 83957, CNRS-INSU/IRD UM 110, Franceb University of Perpignan Via Domitia, CEntre de Formation et de Recherche sur les Environnements Méditerranéens (CEFREM), UMR 5110, 66860 Perpignan, Francec Laboratoire d’Océanographie de Villefranche, UMR 7093, CNRS/UPMC, BP 8, 06238 Villefranche-sur-Mer, Franced Utrecht University, Department of Earth Sciences, PO Box 80.021, 3508 TA Utrecht, The Netherlandse University of Groningen, Arctic Center, PO Box 716, 9700 AS Groningen, The Netherlandsf CSIRO Marine and Atmospheric Research and CSIRO Wealth from Oceans National Research Flagship, GPO Box 1538, Hobart, Tasmania 7001, Australia

a r t i c l e i n f o a b s t r a c t

Article history:Received 19 December 2013Received in revised form 15 May 2014Accepted 4 June 2014Available online 18 June 2014

Keywords:Mackenzie shelfMackenzie RiverSPMSterolsDehydroabietic acidAbiotic and biotic degradationTerrigenous organic matterAutoxidationBacterial degradation

Sterols and their biotic and abiotic degradation products were quantified in suspended particulate matter(SPM) from surface waters in the Mackenzie River mouth to the Beaufort Sea shelf (Canadian Arctic).24-Ethylcholesterol (sitosterol) and 24-methylcholesterol (campesterol) appeared to be extensivelydegraded by bacterial and especially autoxidative degradation in the samples. Degradation was mostextensive in some samples from the outer boundaries of the plume, which exhibited much highersitosterol/campesterol ratio values than previously observed in studies of the Beaufort Sea. The lack ofreactivity of specific planktonic sterols such as cholesterol, 24-methylcholesta-5,22E-dien-3b-ol(epi-brassicasterol) and 24-methylenecholesterol and the good correlation between the abundances ofsitosterol, campesterol and dehydroabietic acid (DHAA, a biomarker of Pinaceae resin) oxidation productsallowed us to attribute the main origin of these two sterols to terrigenous vascular plants. A good corre-lation was observed between the extent of autoxidation and salinity, suggesting that the free radical oxi-dation is enhanced via contact with seawater. Laboratory incubation of Mackenzie River SPM in Milli-Qwater and seawater confirmed this proposal. To explain the specific induction of autoxidation on vascularplant-derived material, a mechanism involving homolytic cleavage of photochemically produced hydro-peroxides resulting from the senescence of higher plants on land is proposed. Cleavage could be catalyzedby redox-active metal ions released from SPM in the mixing zone of riverine water and marine water. Thegreatest extent of degradation observed at outer boundaries of the plume is attributed to preferential set-tling of lithic material relative to less dense higher plant debris increasing the proportion of highlydegraded vascular plant material in the SPM. The results are important for this ecologically vulnerableregion, where destabilization of permafrost by global warming is expected to increase the input of terrig-enous C to coastal seas. Autoxidation, which until now has received little attention, plays a key role in thedegradation of vascular plant-derived lipids in surface waters and should be taken into considerationduring future studies of terrigenous organic matter degradation.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Sterols are valuable biomarkers for assigning the sources oforganic matter (OM) in seawater and sediments (e.g. Gagosianet al., 1982; Brassell et al., 1983; Volkman, 1986). Vascular plants

typically contain simple mixtures of sitosterol (24-ethylcholester-ol), stigmasterol (24-ethylcholesta-5,22E-dien-3b-ol) and campes-terol (24-methylcholesterol), and these three sterols can often beused to trace terrigenous OM in lacustrine systems (e.g. Meyersand Ishiwatari, 1993). In marine systems an additional microalgalinput of these sterols has however, also been identified, soadditional evidence, either from stable isotope signatures (e.g.Matsumoto et al., 2001) or correlation with other plant biomarkers,must be used (e.g. Volkman et al., 2000). Specific sterol degradationproducts have also been proposed for distinguishing biotic from

62 J.-F. Rontani et al. / Organic Geochemistry 75 (2014) 61–73

abiotic processes and photooxidation from autoxidation (Rontani,2008; Rontani et al., 2009, 2011).

The fate of terrigenous OM in the ocean has intrigued scientistsfor decades and is a necessary key for understanding the global Ccycle and its anthropogenic perturbation (Hedges et al., 1997).Because riverine particulate OM (POM) consists in part of alreadyhighly degraded residues from terrigenous higher plants (with ahigh content of lignin), it is generally considered to be refractorywith respect to further decomposition in the ocean (e.g. deLeeuw and Largeau, 1993; Wakeham and Canuel, 2006). Ittekkotet al. (1992) estimated that ca. 35% of the riverine particulateorganic carbon (POC) belongs to the labile (‘‘metabolizable’’) frac-tion and may already be oxidized in estuaries and coastal environ-ments, whereas the other 65% appears to be highly refractory andis therefore preserved in the marginal sediments or transportedfurther offshore. It is thus surprising that only a small fraction ofthe OM preserved in marine sediments appears to be land-derived(Hedges and Keil, 1995). This suggests that either global budgetsand distribution estimates are in error or that POM of terrigenousorigin undergoes rapid and extensive remineralization at sea(Hedges et al., 1997). Indeed, several recent studies have demon-strated that, under some oceanographic conditions, POM deliveredby rivers may be sensitive to microbial remineralization in shelfareas (Mayer et al., 2008; van Dongen et al., 2008; Karlsson et al.,2010; Vonk et al., 2010; Bourgeois et al., 2011).

The Arctic Ocean contains 20% of the Earth’s continental shelfarea (Macdonald et al., 1998) and receives input from many riverswhose collective drainage area contains half of the global soil Cinventory (Dixon et al., 1994; Tarnocai et al., 2009). The MackenzieRiver constitutes the largest fluvial source of both sediment(125 � 109 kg/yr) and POC (2.1 � 109 kg/yr) to the Arctic Ocean(Rachold et al., 2004).

It is thought that the Arctic should provide the earliest and mostdramatic manifestations of global change and that the seasonallyice-covered Arctic Ocean would be one of the first environmentsshowing such an impact (Stroeve et al., 2007). The expected desta-bilization of permafrost and its consequences for hydrology andplant cover is expected to increase the input of terrigenous C tocoastal seas (Benner et al., 2003). Consequently, the relative impor-tance of the fluxes of terrigenous OC and marine OC to the seafloorwould likely change, having a strong impact on the preservation ofOC in Arctic marine sediments (Stein and Macdonald, 2004; Katsevet al., 2006). Before we can predict how global change might influ-ence the delivery and preservation of OC over the Arctic shelves, amore complete understanding of the fundamental processes thatcontrol the degradation and preservation of terrigenous OM isrequired.

Although less widely studied than its biologically mediated(heterotrophic) counterpart, abiotic degradation via processes suchas photooxidation and autoxidation (spontaneous free radicalreaction of organic compounds with oxygen) is now understoodto play a role in the fate of POM in the ocean (Rontani, 2008).Due to the presence of chlorophyll, an efficient photosensitizer(Foote, 1976), visible light-induced photosensitization is intenseduring the senescence of phototrophic organisms (Rontani,2008). This involves mainly singlet oxygen (1O2) and acts on theunsaturated lipid components [including sterols, unsaturated fattyacids (FAs) and the phytyl side chain of chlorophyll] to produceallylic hydroperoxides. The mechanism by which autoxidation isinitiated, debated for many years, seems to be homolytic cleavageof photochemically produced hydroperoxides in phytodetritus(Rontani et al., 2003). Consequently, both photooxidation andautoxidation can significantly affect the composition of lipids inPOM (Rontani, 2008).

Although terrigenous OM is relatively well preserved in sedi-ments from the mid-shelf and shelf edge of the Mackenzie and

the Canada basin of the Beaufort Sea (Yunker et al., 1995, 2005;Belicka et al., 2004), probably via close association with larger mac-romolecules (biopolymers, protokerogen) or detrital mineralgrains, a significant part of this material is likely to be degradedin the water column. To test this, we examined the lipid contentof SPM samples collected in August 2009 off the Mackenzie Riverand surface water of the adjacent Beaufort Sea, in addition toSPM samples from the main branch of the Mackenzie River duringthe spring flood in June 2011. Using specific sterol degradationproducts we have evaluated the roles played by heterotrophic,photodegradative and autoxidative processes in POM degradation.

2. Material and methods

2.1. Study region and sampling

The Mackenzie River provides the largest input of fluvial sedi-ment to the Arctic Ocean, delivering ca. 128 Mt to the MackenzieDelta annually (Carlson et al., 1998). Most of the sediment is deliv-ered during May-October, with peak delivery during the springbreakup and during (local) rainstorm-associated floods in summerand autumn (Brunskill, 1986). It is estimated that 50–63% of theannual sediment load (Carlson et al., 1998; Macdonald et al.,1998) is deposited in the delta. Most of the remaining sedimentload is transported through the delta channels towards ShallowBay and Kugmallit Bay, emptying onto the Mackenzie Shelf. Thelatter is the broadest and most terrigenous dominated shelf alongthe North American coast (Yunker et al., 2005).

During the MALINA cruise in August 2009 (Forest et al., 2013),POM samples were collected (Fig. 1) by filtration of seawater alongtwo transects: one from the Reindeer Channel of the outer Mac-kenzie River Delta to the Mackenzie Canyon and another fromKugmallit Bay to the slope.

2.1.1. Collection of SPM samplesSPM from the Beaufort Sea at stations 690, 680, 620, 380, 360,

340 and 320 (Fig. 1) was collected at 3 m depth using a CTD rosetteequipped with 12 l Niskin bottles, and transferred to glass bottles.Samples of surface water at stations 696, 695, 694, 396, 394 and392 (Fig. 1) were directly collected at 0.5 m depth in glass bottlesfrom a barge. Glassware used for water sample collection wasrinsed with 5% HCl and precombusted at 450 �C for 6 h beforeuse to ensure lack of contamination. Subsamples of 250 to1000 ml water (depending on particle concentration) were filteredimmediately after collection through a precombusted glass fiberfilter (Whatman GF/F, 0.7 lm) under low vacuum to avoid damageto phytoplankton cells. After filtration, the filters were frozenimmediately at �20 �C until analysis and transported to thelaboratory.

Collection of SPM from the Mackenzie River for the incubationexperiments was on June 5, 2011, ca. 1 week after the peak inthe spring flood, near Tsiigehtchic (67.452�N, 133.759�W; Fig. 1).Although the 2011 spring flood SPM may have had a different com-position and abundance from that in August 2009, it was expectedthat both sets of particles would contain highly photooxidizedhigher plant material and a similar complement of metal ionsneeded for autoxidation induction. Surface water (ca. 0.5 m) wascollected from the middle of the channel and transported to thelaboratory at 4 �C, where the water was filtered through 0.7 lmGF/F (Whatman) filters. The filters were kept frozen (�20 �C) untilanalysis.

2.1.2. Collection of water samplesSeawater for incubation experiments was collected from the

northwestern Mediterranean (43.234�N, 5.284�E) using Niskin

Fig. 1. Map of study area with locations of the different stations.

J.-F. Rontani et al. / Organic Geochemistry 75 (2014) 61–73 63

bottles equipped with Teflon O rings and silicon tubes. This samplewas chosen on the basis that its salinity (38‰) was higher thanthat of the Beaufort Sea (31‰) in order to amplify the expected roleplayed by salinity in the induction of autoxidation and to reducethe duration of incubation. Following collection, the water was fil-tered through 0.2 lm polycarbonate filters (Nucleopore, 47 mmdiameter), which were washed with a few ml of 1 M HCl (Merck,NormaPur), 1.5 l of Millipore Milli-Q water and 50 ml of sampleprior to filtration. To try to limit the induction of bacterial activityduring the experiments, filtered seawater was autoclaved for20 min at 100 �C and stored at 4 �C in the dark until the experi-ments were carried out.

2.2. In vitro simulation of suspended particle autoxidation

Incubation experiments were conducted in duplicate. Afterremoval from the GF/F filters by scraping and homogenization,wet SPM from the Mackenzie River (30 mg) was diluted with either100 ml seawater or Millipore Milli-Q water in glass bottles previ-ously rinsed with 5% HCl (Merck NormaPur) and heated at 450 �Cfor 6 h. The solutions were incubated in the dark at room temper-ature (to try to accelerate the processes since autoxidation rateincreases with temperature), using magnetic stirring for 15 days.At the end of the experiments, the samples were filtered onprecombusted (450 �C, 6 h) glass fiber filters (Whatman GF/F,0.7 lm) under low vacuum. The filters were frozen immediatelyat �20 �C until analysis. The incubations could not be carried outunder sterile conditions since heating could destroy photochemi-cally produced hydroperoxides initially present in the particlesand the homolytic cleavage of these during incubation is essentialfor the induction of autoxidation. The use of poison (e.g. HgCl2) wasalso avoided since this could interfere (positively or negatively) inthe initiation of the radical oxidation. The specificity of the tracersemployed (notably steratriols) allowed us to circumvent this prob-lem and to unambiguously attribute the changes to autoxidation.

2.3. Chemical treatment of samples

In situ and in vitro samples were reduced with NaBH4 and sapon-ified. NaBH4 reduction of hydroperoxides to alcohols amenableto gas chromatography–electron ionization mass spectrometry(GC–EIMS) is essential for estimating the importance of photooxida-tive degradation and autoxidative degradation in natural samples

(Marchand and Rontani, 2001). Without this preliminary treatment,these labile compounds can be thermally cleaved during alkalinehydrolysis or GC analysis and thus be overlooked during conven-tional organic geochemical studies. Lipids and their degradationproducts in the resulting total lipid extracts were then quantifiedusing GC–EIMS. All manipulations were carried out with foil cov-ered vessels in order to exclude photochemical artifacts. It is wellknown that metal ions can promote autoxidation during hot sapon-ification (Pokorny, 1987). The prior reduction of hydroperoxideswith NaBH4 allowed us to avoid such autoxidation artifacts duringalkaline hydrolysis.

Filters were placed in MeOH (15 ml) and hydroperoxides werereduced to the corresponding alcohols with excess NaBH4 (70 mg;30 min at 20 �C). During the treatment, ketones are also reducedand the possibility of some ester cleavage cannot be excluded.

Saponification was carried out on reduced samples. After NaBH4

reduction, 15 ml water and 1.7 g KOH were added and the mixturedirectly saponified by refluxing for 2 h. After cooling, the content ofthe flask was acidified with HCl (to pH 1) and extracted (3�) withdichloromethane (DCM). The combined DCM extracts were driedover anhydrous Na2SO4, filtered and concentrated to give the totallipid extract. After solvent evaporation, the residue was taken up in300 ll pyridine/N,O-bis(trimethysilyl)trifluoroacetamide (BSTFA;Supelco; 2:1, v:v) and silylated for 1 h at 50 �C to convert OH-containing compounds to TMSi ether or ester derivatives.

After evaporation to dryness under a stream of N2, the deriva-tized residue was taken up in a mixture of EtOAc/BSTFA (to avoiddesilylation of FAs) for analysis using GC–EIMS. It should be notedthat, under these conditions, steran-3b,5a,6b-triols are silylatedonly at C-3 and C-6 and thus need to be analyzed with care. Theuse of hot splitless injectors (which can discriminate against highboiling compounds and induce thermal degradation) should beavoided. The best results were obtained with an on-columninjector coupled to a deactivated retention gap. The use of morepowerful silylating reagents such as N-trimethylsilylimidazole/N,O-bis(trimethylsilyl)acetamide/trimethylchlorosilane (TMSIM/BSA/TMCS) (Bortolomeazzi et al., 1999) or BSTFA/dimethylsulfox-ide yields complete silylation of 3b,5a-dihydroxysterols. Unfortu-nately, the presence of an additional (easily silylated) 6b-OH insteran-3b,5a,6b-triol compounds induces additional steric hin-drance, which precludes silylation at C-5.

Compounds were assigned by comparison of retention times andmass spectra with those of standards and quantified (calibration

64 J.-F. Rontani et al. / Organic Geochemistry 75 (2014) 61–73

with external standards) with GC–EIMS. For low concentrations, orin the case of co-elution, quantification was achieved using selectedion monitoring (SIM). The main characteristic fragment ions used toquantify degradation products of sterols have been described byChristodoulou et al. (2009) and Rontani et al. (2011).

GC–EIMS was carried out using an Agilent 6890 gas chromato-graph connected to an Agilent 5973 Inert mass spectrometer. Theconditions were: 30 m � 0.25 mm (internal diameter) fused silicacolumn coated with SOLGEL-1 (SGE; 0.25 lm film thickness); oventemperature 70 �C to 130 �C at 20 �C/min, then to 250 �C at 5 �C/min and then to 300 �C at 3 �C/min; carrier gas (He) 0.69 �105 Pa until the end of the temperature program and then from0.69 � 105 Pa to 1.49 � 105 Pa at 0.04 � 105 Pa min�1; injector(splitless), 250 �C; injector (on column), 50 �C; electron energy,70 eV; source, 170 �C; cycle time, 1.5 s. An on-column injectorwas used for analysis of sterol degradation products and a splitlessinjector for analysis of FA degradation products. Analysis in SIMmode allowed detection of degradation products with a detectionlimit of 1–10 ng (depending on compound injected). Standard oxi-dation products of sterols were obtained according to describedprocedures (Rontani and Marchand, 2000).

2.4. POC analysis

The concentration of POC in mg/l in each water sample wasmeasured using a pre calibrated Perkin Elmer 2400 Series IICHNS/O analyzer, where samples are combusted in a pure O2 envi-ronment, with the resultant combustion gases measured with athermal conductivity detector. Prior to combustion, filters wereacidified with 2 N HCl (0.2 ml, followed by further additions of0.15 ml until no more fizzing was observed) to remove carbonateand then dried overnight at 60 �C before analysis (King et al.,1998). The blank filters were also acidified and dried overnight.Laboratory experiments conducted with the CHN analyzer beforeand after the MALINA cruise showed that POC measurementshad a typical uncertainty of ± 4%.

Fig. 2. D5-Sterol degradation products used to estimate biodegradation, autoxidation aet al., 2009).

2.5. Lipid degradation products employed for photooxidation,autoxidation and biodegradation estimation

1O2 mediated photooxidation of D5-sterols produces (Fig. 2)mainly D6-5a-hydroperoxides with smaller amounts of D4-6a/b-hydroperoxides (Kulig and Smith, 1973). The latter were selectedas tracers of photooxidation of D5-sterols due to their high specific-ity and relative stability (Christodoulou et al., 2009; Rontani et al.,2011). As the hydroperoxides could not be analyzed directly, theywere quantified after NaBH4 reduction to the corresponding diols,and the sterol photooxidation proportion was estimated using Eq.1 (Christodoulou et al., 2009), based on the ratio of 0.30 for D4-6a/b-hydroperoxides/D6-5a-hydroperoxides in biological membranes(Korytowski et al., 1992).

Sterol photooxidation % ¼ ðD4-3b;6a=b-dihydroxysterol %Þ� ð1þ 0:3Þ=0:3 ð1Þ

Free radical autoxidation of D5-stenols yields (Fig. 2) mainly7a- and 7b-hydroperoxides and, to a lesser extent, 5a/b,6a/b-epoxysterols and 3b,5a,6b-trihydroxysterols (Smith, 1981). Weselected 3b,5a,6b-trihydroxysterols as tracers of sterol autoxida-tion. 7-Hydroperoxides were ruled out as possible markers dueto their lack of specificity and instability (Christodoulou et al.,2009) and 5a/b,6a/b-epoxysterols could not be used since theyare converted to the corresponding triol during saponification.The extent of sterol autoxidation was estimated using Eq. 2 basedon autoxidation rate constants obtained from our incubationexperiments (k7-OOH/ktriol = 0.60) and calculated by Morrissey andKiely (2006) (k7-OOH/kepox = 0.85). An averaged value of 0.725 wasused during the calculations.

Sterol autoxidation % ¼ ð3b;5a;6b-trihydroxysterols %Þ� ð1þ 0:725Þ=0:725 ð2Þ

Complete mineralization of D5-sterols may be achieved by bacteriafrom several genera (Owen et al., 1983; Naghibi et al., 2002) and

nd photooxidation state of POM (adapted from Christodoulou et al., 2009; Rontani

J.-F. Rontani et al. / Organic Geochemistry 75 (2014) 61–73 65

they can also undergo aerobic bacterial reduction, thought to be awidespread process (Fig. 2) in the marine environment and leadingmainly to ster-4-en-3-ones, 5a(H)-stanones and 5a(H)-stanols(Gagosian et al., 1982; de Leeuw and Baas, 1986; Wakeham,1989). These steroid ketones and stanols thus constitute usefulindicators of bacterial degradation of stenols. During NaBH4 reduc-tion, ster-4-en-3-ones and 5a(H)-stanones were converted to ster-4-en-3-ols and 5a(H)-stanols respectively (Fig. 2).

The use of these different tracers to estimate the relative impor-tance of the degradation processes requires that the removal rates(via further degradation) are similar to those of the parent D5-ster-ols. Although each sterol and its degradation products may bepotentially totally mineralized by marine bacteria, we assume thatthey should exhibit similar reactivity towards bacterial degrada-tion. The assumption is based on the fact that aerobic biodegrada-tion of sterols generally involves initial attack on the side chain,which is similar for all the degradation tracers selected to that ofthe corresponding parent D5-sterol.

3. Results

3.1. Degradation of sterols along the two transects

Sterol profiles of most of the samples were dominated by choles-terol and 24-ethylcholesterol (sitosterol if the C-24 stereochemistryis 24a). 24-Methylcholesta-5,22E-dien-3b-ol (brassicasterol and/orepi-brassicasterol depending on C-24 stereochemistry), 24-methyl-cholesterol (campesterol if the C-24 stereochemistry is 24a),24-methylcholesta-5,24(28)-dien-3b-ol (24-methylenecholesterol)and 24-ethylcholesta-5,22E-dien-3b-ol (stigmasterol if the C-24stereochemistry is 24a) were also detected but in lesser amount.The abundances of sitosterol (0.02–1.9 lg/l) and cholesterol(0.26–2.5 lg/l) (Table 1) were different from those measured by

Table 1Concentrations of sterols, DHAA and their degradation products (lg/l), SPM and POC at di

Compounds Station396

Station394

Station392

Station380

24-Ethylcholest-5-en-3b-ol 0.13 0.10 0.12 0.9824-Ethyl-5a(H)-cholestan-3b-ol 0.03 0.03 0.02 1.6824-Ethylcholest-4-en-3b,6a/b-diols – – – 0.2924-Ethylcholestan-3b,5a,6b-triol 0.06 0.08 0.06 10.20

24-Methylcholest-5-en-3b-ol 0.01 0.03 0.01 0.0324-Methyl-5a(H)-cholestan-3b-ol 0.01 0.03 0.01 0.0624-Methylcholest-4-en-3b,6a/b-diols 0.01 – – –24-Methylcholestan-3b,5a,6b-triol 0.01 0.03 0.01 0.28

Cholest-5-en-3b-ol 0.46 0.57 0.33 0.415a(H)-Cholestan-3b-ol 0.01 0.03 0.01 0.01Cholest-4-en-3b,6a/b-diols 0.01 – 0.01 –Cholestan-3b,5a,6b-triol 0.01 0.03 0.01 0.01

24-Methylcholesta-5,22E-dien-3b-ol 0.21 0.10 0.03 0.0624-Methyl-5a(H)-cholest-22E-en-3b-ol – – – –24-Methylcholesta-4,22E-dien-3b,6a/b-diols – – – –24-Methylcholest-22E-en-3b,5a,6b-triol – – – –

24-Methylcholesta-5,24(28)-dien-3b-ol 0.07 0.06 0.02 0.0324-Methyl-5a(H)-cholest-24(28)-en-3b-ol – – – –24-Methylcholesta-4,24(28)-dien-3b,6a/b-diols – – – –24-Methylcholest-24(28)-en-3b,5a,6b-triol – – – –

(R Sterols + degradation products)/POC (%) 0.7 04 0.5 10.0Sitosterol/campesterolb 6 2 7 36

DHAA 0.16 0.14 0.11 2.61DHAA degradation products 0.01 0.01 0.01 0.86Water depth to sediment (m) 6 14 27 60SPM (mg/l) 2.00 3.40 0.52 0.40POC (mg/l) 0.14 0.26 0.12 0.14POC/SPM (%) 7.0 7.7 23.1 35.0

a Proportion < 0.01.b Ratio (R sitosterol + its degradation products)/(R campesterol + its degradation prod

Yunker et al. (1995) in SPM from the Mackenzie Shelf in spring (val-ues reaching 0.12 and 6.4 lg/l for sitosterol and cholesterol, respec-tively). Concentrations of sterols appeared to be significantly higherin POM from the Mackenzie Shelf (24–2450 lg/g for sitosterol) thanin sediments from the same zone (0.5–13.9 lg/g, Rontani et al.,2012a; 0.8–8.0 lg/g, Yunker et al., 1995). The lower sterol concen-tration in sediments is likely caused by efficient degradation duringboth water column scavenging and lateral bottom transport of par-ticles. The assumption is supported by the strong photodegradationstate of sinking particles, which are generally considered as themain contributors to the sedimentary record (Wakeham and Lee,1989) in the Mackenzie Shelf (Rontani et al., 2012b). The proportionof sterols strongly increased at stations 380, 690 and 620 (Table 1,Fig. 3). These changes likely result from intense bacterial minerali-zation of the more labile OM components (including the straightchain lipids; Atlas and Bartha, 1992) in seawater.

Samples from stations 620, 690 and 380 (Mackenzie Canyon andKugmallit Valley, respectively) contained a high proportion of sitos-terol degradation products (Table 1, Fig. 3b). Sitosterol is present insome marine microalgae (Volkman, 1986, 2003), but a vascularplant origin typically dominates in areas strongly influenced by ter-rigenous sources (Yunker et al., 1995; Belicka et al., 2004). Sitosterolcomprises up to 95% of the total sterols in plants (Lütjohann, 2004)and it is abundant in peat known to be transported to this region(Yunker et al., 1995). In sediments of the Beaufort Sea, it was estab-lished that sitosterol results mainly from terrigenous material(Goñi et al., 2000; Belicka et al., 2004). The presence of a highproportion of 24-ethyl-5a(H)-cholestan-3b-ol at stations 620, 690and 380 (Table 1, Fig. 3b) confirmed the important role played bybacterial degradation in the fate of sitosterol (Fig. 2). Small amountsof 24-ethylcholest-5-en-3b,26-diol and 24-ethyl-5a(H)-cholestan-3b,26-diol were also detected in these samples (Fig. 3b). Since thebacterial degradation of sterols generally starts with terminal

fferent stations.

Station360

Station340

Station320

Station696

Station695

Station694

Station690

Station680

Station620

0.04 0.12 0.02 1.84 0.54 0.15 0.54 0.46 0.280.01 0.02 0.01 0.19 0.06 –a 0.54 0.11 2.02– 0.01 – 0.10 – – 0.07 0.11 0.280.01 0.06 0.01 0.39 0.18 0.06 2.69 0.29 12.91

0.01 0.01 0.01 0.19 0.03 0.03 0.02 0.13 0.010.01 0.01 – 0.10 0.03 – 0.01 0.06 0.06– 0.01 – – – – – 0.02 0.01– 0.01 – 0.10 – – 0.07 0.04 0.34

0.35 0.26 0.71 2.52 0.36 0.28 0.36 0.46 1.000.02 0.01 0.02 0.10 0.03 0.03 0.01 0.06 0.020.01 0.01 0.01 – – – – 0.02 0.010.01 0.01 0.01 0.10 0.03 – 0.01 0.02 0.03

0.01 0.02 0.01 0.19 0.15 0.16 0.02 0.17 0.01– – – – – – – 0.02 –– – – – – – – – –– – – – – – – – –

0.01 – 0.01 – 0.03 0.03 0.01 0.06 0.01– – – – – – – – –– – – – – – – – –– – – – – – – – –

0.8 0.9 1.4 0.6 0.4 0.3 4.0 1.1 23.83 5 4 7 13 8 38 4 37

0.18 0.28 0.20 6.72 1.91 1.61 1.72 2.64 2.340.01 0.01 0.01 0.12 0.05 0.05 0.22 0.08 1.0275 553 1140 3 5 8 40 105 15380.36 0.18 0.25 82.1 20.6 10.7 1.27 0.92 0.340.06 0.06 0.06 0.97 0.30 0.31 0.11 0.19 0.0716.7 33.3 24.0 1.2 1.5 2.9 8.7 20.7 20.6

ucts).

Fig. 3. Total ion current chromatograms from total lipid extracts from stations 695 (a) and 620 (b) (� n-alkanoic acids; * n-alkanols; Sq, squalene; Ph, phthalate).

66 J.-F. Rontani et al. / Organic Geochemistry 75 (2014) 61–73

attack of the side chain (Owen et al., 1983), these diols likely repre-sent early bacterial metabolites of sitosterol.

While strong photooxidation of phyto- and zooplanktonic mate-rial was recently observed in sinking particles collected with sedi-ment traps in summer in the same zone (Rontani et al., 2012b),24-ethylcholest-4-en-3b,6ab-diols, specific tracers of photooxida-tion, were only detected in small amounts in the samples investi-gated (Table 1). This is probably due to an intense free radicaldriven breakdown of photochemically produced hydroperoxides(Rontani et al., 2003). The assumption is well supported by theresults from laboratory incubations (Section 3.2). Indeed, the pres-ence of large amounts of 24-ethylcholestan-3b,5a,6b-triol in someof the samples (Table 1, Fig. 3b) indicates that autoxidation plays animportant role in the degradation of sitosterol (Fig. 2). In the case ofthe Mackenzie Canyon transect, the proportion of sitosterol autox-idation products (estimated from the triol abundance using Eq. 2)was dramatically higher at station 620 at the end of the canyon(Fig. 4d), while in the other transect it maximized at station 380(Kugmallit Valley) (Fig. 4a) and then decreased (probably due todilution of the terrigenous signal by marine material) in the slopewaters. This hypothesis is well supported by the d13C values mea-sured by Retamal et al. (2007) for POM samples from stations 380(�30.6‰) and 320 (�24.9‰).

Similar results to sitosterol were obtained for campesterol(Table 1, Fig. 4b and e), which is also a component of vascular plants(Lütjohann, 2004). In contrast, cholesterol, epi-brassicasterol and24-methylenecholesterol, which are derived mainly from plank-tonic sources (Volkman, 1986, 2003), appeared to be much lessaffected by biotic and abiotic degradation (Table 1, Fig. 4c and f).

3.2. Laboratory incubation experiments

SPM collected in June 2011 from the Mackenzie River was sha-ken (with magnetic stirring) in seawater and Milli-Q water underdarkness at room temperature for 15 days. Vascular plant-derivedsitosterol was initially highly photodegraded in these particles.Indeed, the proportion of photodegradation products relative to

the residual parent compound was estimated using Eq. 1 to be closeto 50% (Fig. 5a). After incubation in Milli-Q water, a slight but signif-icant production of 24-ethylcholestan-3b,5a,6b-triol was observed(Fig. 5a), attesting to the involvement of autoxidation. Althoughautoxidation also produces 7a- and 7b-hydroperoxides (Fig. 2),the amount of 24-ethylcholest-5-en-3b,7a/b-diols (obtained afterNaBH4 reduction) decreased during incubation (Fig. 5a). Thedecrease is attributed to a free radical driven breakdown of the cor-responding hydroperoxides. This assumption is supported by thedecreasing amount of 24-ethylcholest-4-en-3b,6a/b-diols (specificphotooxidation products not produced under darkness; Fig. 2), alsoobserved during incubation (Fig. 5a). The sterol/stanol ratio(indicative of bacterial degradation), which remained practicallyunchanged during incubation, attested to a lack of intensive biodeg-radation under the conditions. It is interesting to note, however,that in seawater the production of 24-ethylcholestan-3b,5a,6b-triolwas strongly enhanced, while the amounts of 24-ethylcholest-4-en-3b,6a/b-diols and 24-ethylcholest-5-en-6b,7a/b-diols stronglydecreased (Fig. 5a). As proposed in the previous section, the verysmall amounts of photooxidation tracers of vascular plant-derivedsterols in SPM samples from the Beaufort Sea therefore seemed toresult from a free radical driven breakdown of these compoundsin seawater. Incubation in Milli-Q water and seawater had littleeffect on cholesterol abundance (Fig. 5b), which arises mainly fromplanktonic organisms and was initially weakly photodegraded inSPM from the Mackenzie. Indeed, this sterol appeared to be autox-idized to a lesser extent (6%) than sitosterol (45%) after incubationin seawater (Fig. 5b). These results confirm the important roleplayed by the initial photooxidation state of detrital particles(higher plant debris or phytodetritus) in the induction of autoxida-tion (Rontani et al., 2014).

3.3. Degradation of other terrigenous biomarkers

All the samples contained a significant amount of dehydroabiet-ic acid (8,11,13-abietatrien-18-oic acid; DHAA). This compound,often used as biomarker of coniferous residues in sediments

Fig. 4. Salinity (diamonds) and estimates of relative extents of biodegradation, autoxidation and photooxidation (%) of sitosterol (A and D), campesterol (B and E) andcholesterol (C and F) along the two transects.

J.-F. Rontani et al. / Organic Geochemistry 75 (2014) 61–73 67

(Brassell et al., 1983) and biomass burning (Oros and Simoneit,2001; Simoneit, 2002), is present as a minor component of thefresh resin of conifers, but its abundance increases on ageing atthe expense of the corresponding abietadienic acids. If oxygen isavailable, DHAA can be oxidized to 7-oxodehydroabietic acid, 15-hydroperoxyabietic acid and numerous other degradation products(Shao et al., 1995; Martin and Mohn, 2000). 7-OxoDHAA and twoepoxides deriving from homolytic cleavage of 7- and 15-hydroper-oxyDHAA (Gälvert et al., 1994) could be detected as TMS ethers inthe extracts. The values of the ratio DHAA/(7-oxoDHAA + epoxy-DHAA) are given in Fig. 6. The most extensive state of degradationof DHAA was observed at stations 620, 690 and 380.

The dihydroxylated triterpenoid, betulin [lup-20(29)-ene-3b,28-diol], also of plant origin, was also present in most of thesamples, but in highly variable proportion. Its biotic and abioticdegradation products are presently unknown.

4. Discussion

4.1. Source of OM in SPM of the Mackenzie River

Yunker et al. (1995) observed that the OM in SPM from theMackenzie River was composed of contributions from bacteria,microalgae and vascular land plants. More recently, Tolosa et al.(2013) estimated that algal and C3 vascular plant contributionswere 55 and 10% of total OM, respectively, and concluded thatthe algal material was dominated by diatoms. The Mackenzie Riverflows through various types of land, including Arctic tundra, borealforest, peatland and mountains (Dyke and Brooks, 2000). Tundra isgenerally considered an ecosystem rather decoupled from the river

system, while the boreal forest, mostly conifers, constitutes animportant source of terrigenous OM of higher plant origin for thisriver (Solomon et al., 2000; Carrie et al., 2009). Although lignin andd13C data have indicated that the major source of terrigenousmaterial in this area consists of non-woody, C3 angiosperm vascu-lar plant vegetation (Goñi et al., 2000), the recent detection of rel-atively enriched d13C values for n-alkanes and n-alkanols suggesteda significant contribution from gymnosperms (Tolosa et al., 2013).This contribution is consistent with the presence of significantamounts of DHAA (0.7 lg/l) in our Mackenzie SPM sample. TheMackenzie River also contains fossil material, such as shale or coalfragments (Yunker et al., 2002, 2011).

4.2. Sterol degradation

The highest contents of sitosterol and campesterol degradationproducts (stanols and triols) at stations 620, 690 and 380 (ca. oneorder of magnitude higher than at the other stations; Table 1,Fig. 4) could be attributed to preferential settling of lithic (min-eral-bound) material relative to less dense higher plant debris(Yunker et al., 1995; Dittmar and Kattner, 2003), which may havestrongly increased the relative proportion of highly degraded vas-cular plant material in suspended OM of the stations at outerboundaries of the plume (as for stations 620 and 380 in summer).Indeed, the river transports a mixture of plant detritus, soil andlithic material that is introduced to the river primarily via snow-melt and episodic rainfall events (Yunker et al., 2002), and is wellmixed prior to leaving the delta. One of the main processes affect-ing this well mixed material on the shelf is the settling of lithicmaterial (Yunker et al., 1995). This hypothesis is supported bythe contribution of POC to SPM, which increases from 7.6% at

Fig. 5. Quantification of autoxidation products of sitosterol (a) and cholesterol (b)before and after incubation of Mackenzie River suspended particulate matter (SPM)in seawater and Milli-Q water under darkness at room temperature.

Fig. 6. Proportion (%) of degradation products of dehydroabietic acid (DHAA)(relative to residual parent compound) along the two transects.

68 J.-F. Rontani et al. / Organic Geochemistry 75 (2014) 61–73

station 394 to 35.6% at station 380 and from 1.2% at station 696 to20.6% at station 620 (Table 1) and by a similar very strong oxida-tion state for sitosterol and campesterol observed in two othersamples collected at the eastern boundaries of the surface plumefrom the river runoff (70.55037�N 128.55132�W and 70.52166�N130.30041�W) (Rontani, data not shown). The low degradationstate of specific planktonic sterols (brassicasterol and 24-methy-lenecholesterol; Volkman, 1986, 2003) observed at the stationscorresponding to the outer boundaries of the plume allowed usto exclude a significant contribution of marine material to thesetwo highly degraded sterols. Due to the relatively low terrigenousnatural background and sterol content of aerosols collected in thiszone during the sampling period (Fu et al., 2013), a significantatmospheric input of highly degraded sitosterol and campesterolto the samples also seems unlikely. On the basis of a previousexamination of surface sediments from a station very close to sta-tion 690 (Rontani et al., 2012a), showing a relatively low autoxida-tion state for sitosterol and campesterol (< 20% and 10% of theresidual parent compound, respectively), an input of low densityparticles from resuspended sediment can be also discounted.

It is interesting to note that Yunker et al. (1995) explained thehigher odd/even predominance of n-alkanes in Mackenzie Shelfsuspended particles than in Mackenzie River particles or shelf sed-iments to the presence of a higher proportion of low density vascu-lar plant debris. The presence of well preserved terrigenous lipidtracers in sediments from the Beaufort Sea (Yunker et al., 1995;Belicka et al., 2004) could thus be attributed to the rapid settlingof terrigenous OM associated with lithic material. However, the

recent detection of sitosterol and campesterol degradation prod-ucts in surface sediments from this area (Rontani et al., 2012a)indicates that a part of the highly degraded higher plant debris alsoreaches the sediments, probably after aggregation.

While autoxidation appears relatively limited within theMackenzie River itself (see results from initial material duringlaboratory incubations, Fig. 5), the process plays a key role in thedegradation of terrigenous sterols in the Beaufort Sea (Fig. 4). Sig-nificant correlation was observed between salinity and the propor-tion of autoxidation of sitosterol (relative to the parent sterol andits other degradation products; estimated using Eq. 2) from station696 to station 620 (Fig. 4d) (r2 0.75, p = 0.025, n = 6). The correlationwas not significant for samples from transect 300 (Fig. 4a), probablydue to a marine contribution to the sitosterol content of some ofthese samples (Retamal et al., 2007). However, it should be notedthat two SPM samples collected at 0 and 10 m at station 394, closeto the Mackenzie River mouth, where a strong salinity gradient isestablished (21.4‰ at the surface and 30.1‰ at 10 m), exhibitedan autoxidation proportion of 4.8% and 94.1%, respectively. It thusseems that free radical oxidation was induced by contact withseawater. The difference in degradation state between the samplescollected at stations 620 and 690 exhibiting similar salinity (Fig. 4)can be attributed to the residence time of low density vascular plantdebris in seawater (Tolosa et al., 2013), which should be longer atthe 620 station more distant from the Mackenzie River mouth. Itis interesting to note that the strong spatial variability in the autox-idative degradation state of sitosterol and campesterol previouslyobserved in surface sediments of the Mackenzie Shelf (Rontaniet al., 2012a) was also attributed to the position of the stations rel-ative to the Mackenzie River mouth. Indeed, the stations more dis-tant from the river mouth, where the residence time of terrigenousOM in seawater is expected to have been longest, exhibited thehighest autoxidation state.

J.-F. Rontani et al. / Organic Geochemistry 75 (2014) 61–73 69

Laboratory incubation of SPM from the Mackenzie River in mil-liQ water and seawater allowed us to demonstrate unambiguouslythat free radical oxidation of sterols may be enhanced by seawater,but also that it acts mainly on vascular plant-derived sitosterol(Fig. 5). The high reactivity of components of higher plant debristowards free radical oxidation can likely be attributed to their ini-tial photodegradation on land or into the river. Indeed, homolyticcleavage of photochemically produced hydroperoxides may initi-ate free radical oxidation chains (Rontani et al., 2003, 2014).Redox-active metal ions play an important role in this homolysisbecause they are ubiquitous and active in many forms and tracequantities are sufficient for effective catalysis (Schaich, 2005). Onlymetals undergoing one electron transfer appear to be active cata-lysts and include Co, Fe, Cu, Mn, Zn, Mg and V. The metal ionsmay direct the cleavage of hydroperoxides either through forma-tion of alkoxyl or peroxyl radicals. We thus hypothesize that thestrong induction of sitosterol and campesterol autoxidation inthe Beaufort Sea results from the release of metal ions (such asCu2+, Zn2+ and V2+, which have been detected in sediments fromthe Beaufort Shelf; Crecelius et al., 1991) able to catalyze homoly-sis of photochemically produced hydroperoxides from vascularplant-derived POM during the mixing of the Mackenzie waterand marine water.

4.3. Origin of sitosterol and campesterol in the samples

Compound-specific d13C analysis has been carried out on SPMsamples collected from deeper and at different stations of transects300 and 600 and compared with these from the Mackenzie River(Tolosa et al., 2013). Unfortunately, d13C values obtained for mar-ine phytoplanktonic biomarkers synthesized at this high latitudearea appear to have similar d13C values to those of C3 terrigenousbiomarkers, thereby making it problematic to discern the source(s)of sitosterol in the marine SPM based solely on the d13C signature.

The use of correlations between concentrations of biomarkersthat are structurally different to infer a common origin is poten-tially valuable but the effects of differential degradation must alsobe considered. This is notably the case for the dihydroxylated tri-terpenoid betulin, present in most of the samples, and sitosterol.This diagnostic higher plant marker has been found in peat-derivedOM deposited in the Wadden Sea (Volkman et al., 2000). Its pres-ence in the samples is in good agreement with Yunker et al.(1993), who reported the presence of peat material in sedimentsfrom the Beaufort Sea. However, it was demonstrated by Kochet al. (2005) that betulin is degraded much faster than sitosterolin surface sediments. Also, the lack of information on possible deg-radation products of this triterpenoid, makes any correlation of itsabundance with that of sitosterol and campesterol problematic.

In contrast, good correlation was obtained between the degra-dation proportions of sitosterol and DHAA (tracer of Pinaceaeresin; r2 0.98 and 0.96 for transects 600 and 300, respectively,n = 6; Figs. 4 and 6) and between the concentration of (R ste-rol + degradation products)/mg of SPM and (R DHAA + degradationproducts)/mg of SPM (r2 0.94 and 0.99 for sitosterol and campes-terol, respectively, n = 11). Sitosterol, campesterol and DHAA thusseem to have a common terrigenous origin for the samples inves-tigated. Although the involvement of atmospheric input (plant-derived aerosols or biomass burning) in this zone cannot be totallyexcluded, it seems unlikely. Indeed, the contribution of biomassburning and terrestrial natural background to OM of marine aero-sols during the MALINA campaign was recently estimated to beonly 1.1–12.8% (mean 4.3%) and 2.2–6.3% (mean 3.6%), respectively(Fu et al., 2013). These low values could be explained by both wet/dry deposition and atmospheric dilution of aerosol particles duringlong range transport (Fu et al., 2013). Moreover, DHAA is present in

relatively high proportion in the Mackenzie River and the strongoxidation of plant-derived OM was observed in very distant sta-tions all located at the outer boundaries of the plume.

Volkman (1986) suggested that evaluation of campesterol/stig-masterol/sitosterol ratios could be used to determine if these ster-ols are appropriate to be used as terrestrial biomarkers. The valueof the ratio (R sitosterol + its degradation products)/(R campester-ol + its degradation products) for our Mackenzie River SPM sample(6.4) is in good agreement with the values obtained by Yunkeret al. (1995; 5.7 ± 1.5) and confirms the presence in the river of amixture of algal and terrigenous higher plant material. The veryhigh values for stations 380, 620 and 690 (ranging from 36 to38; Table 1) are quite unusual and rarely observed in eithersediments or SPM in different oceanic environments. These sitesare also those where the greatest sitosterol and campesterol degra-dation was observed (Fig. 4), which we have suggested is due tothe strong accumulation of low density vascular plant debris atthese stations. While many plants have a sitosterol/campesterolvalue < 4 (Volkman, 1986), Nishimura (1977) reported values rang-ing from 11.5 (Pinus densiflora) to 31 (holly Ilex pedunculosa). It maybe noted from sterol data measured by Tolosa et al. (2013) for SPMsamples collected at different depths in the Beaufort Sea during theMALINA cruise, that this ratio was highly variable (ranging from 53to 1.5), implying the involvement of several sources. Interestingly,the only value obtained by these authors for near surface (3 m)waters was relatively high (19).

The low ratio values for surface sediment from the MackenzieShelf (5.5 ± 0.9, Yunker et al., 1995; 4.7 ± 1.0, Belicka et al., 2004;3.5 ± 2.0, Rontani et al., 2012a) likely result from contributionsfrom plants, peat and freshwater diatoms to this material. The algalcontribution to OM in surface sediments of the Mackenzie Shelfwas recently estimated by Tolosa et al. (2013) to be 80–90%. Theimportance of diatoms in this algal material is supported by thepresence of significant proportions of 24-methylenecholesterol(diatom marker, Volkman, 1986, 2003) in these sediments (ratiosof sitosterol/24-methylenecholesterol of 0.3–7.0; Yunker et al.,1995; Belicka et al., 2004; Rontani et al., 2012a; Tolosa et al.,2013). It is interesting to note that some sea ice and freshwaterdiatoms exhibit sitosterol/campesterol values ranging from 1.0 to6.8 (Nichols et al., 1990; Ponomarenko et al., 2004).

Moreover, comparison of the degradation state of sitosterol andcampesterol with that of specific phytoplanktonic sterols (e.g. epi-brassicasterol and 24-methylenecholesterol, Volkman, 1986, 2003;Table 1) allowed us to exclude a major contribution from microal-gal sterols to these sterols. Indeed, even though diunsaturatedepibrassicasterol and 24-methylenecholesterol are expected to bemore reactive towards autoxidation (Frankel, 1998) than monoun-saturated sterols, and similarly affected by bacterial degradation(Wakeham and Beier, 1991), they are relatively unaffected by theseprocesses at stations 620, 690 and 380, while sitosterol and cam-pesterol are strongly altered. This contrasting biotic and abioticbehavior suggests distinct sources. These different indicators areall consistent with the view that the major source of sitosteroland campesterol in the samples is terrigenous plants includingconifers.

4.4. Fate of POM in the Mackenzie River and Beaufort Sea

From the abundance of D5-sterols and their degradation prod-ucts in suspended particles collected from the Mackenzie mouthto the Beaufort Slope, we have been able to infer the extent of deg-radation of terrestrial and marine POM in this zone. Using sitos-terol and campesterol as indicators of vascular plant-derived OM,we conclude that biodegradation and autoxidation act intensivelyon terrestrial POM in surface waters of the Beaufort Shelf. These

70 J.-F. Rontani et al. / Organic Geochemistry 75 (2014) 61–73

observations agree with the modeling results of van Dongen et al.(2008) who showed that 73% of sitosterol discharged by the KalixRiver was degraded during its transit through the inner low salin-ity zone during the spring flood, in contrast to the estimates ofIttekkot et al. (1992) concerning the refractory character of terres-trial OM. Some vascular plant biomarkers, which are probablyphysically protected from bacterial degradation by close associa-tion with larger macromolecules (biopolymers, protokerogen) ordetrital mineral grains on land and in the river (Drenzek et al.,2007; Vonk et al., 2010), appear to be quickly mineralized by bac-teria in surface water of the Beaufort Sea.

Denaturing gradient gel electrophoresis analysis has revealedchanges in the structure of bacterial communities associated withparticles from the Mackenzie River to the shelf (Garneau et al.,2008), giving rise to the possibility that the bacterial assemblagein the marine environment can use specific parts of terrestrialPOM more effectively than the bacterial assemblage in soils andrivers. The involvement of ‘priming effects’ (enhanced reminerali-sation of terrestrial OM in the presence of fresh substrates fromalgal sources; Bianchi, 2011) can not be totally excluded. Coastaldeltaic regions, where there is a high input of terrestrial OM, aswell as, in many cases, high primary production, nutrient, light,and salinity gradient and microbial communities highly adaptableto physicochemical changes, are generally considered to have ahigh priming potential (Bianchi, 2011).

Recalcitrant terrestrial OM can be made available for bacterialutilization and assimilation by co-metabolism. Co-metabolism,

Fig. 7. Schematic showing the likely fate of terrestrial OM and marine OM in the Beaufrelative importance of the different degradation processes, which are based on sterol de

defined as ‘‘degradation of a recalcitrant substance by microorgan-isms in the presence of a readily degradable substrate’’ may consti-tute an important mechanism in breaking down recalcitrantmaterial (Canfield, 1994). The high photooxidation state of terrige-nous OM in the Mackenzie River (Section 3.2) may be a fundamen-tal factor driving the intense biodegradation of this materialobserved in seawater. Indeed, Vähätalo et al. (1998) observed thatseagrass exposed to solar light supported 2.1–3.4 times more bac-terial activity than when kept under darkness. Solar light-inducedfragmentation can reduce structural barriers in higher plant debrisand expose new microniches to bacterial colonization (Vähätaloet al., 1998). Moreover, these processes can degrade phenols(Opsahl and Benner, 1993), which are present in fresh leaves ofhigher plants (Zapata and McMillan, 1979) and can inhibit bacterialgrowth (Harrison, 1982).

Autoxidation (spontaneous free radical reaction of organic com-pounds with O2) in the marine realm, which has been largelyignored until now, can act not only on unsaturated lipids (e.g.sterols, unsaturated FAs, chlorophyll phytyl side chain, alkenes,tocopherols and alkenones; Rontani, 2008), but also on amino acids(Seko et al., 2010), sugars (Lawrence et al., 2008) and polyphenols(Hathway, 1957). It can also affect biopolymers (Schmid et al.,2007) and kerogen, inducing ring opening and chain cleavage,which may enhance bacterial degradation of these recalcitrant sub-strates (Bianchi, 2011). These degradation processes, which seem tobe catalyzed by metal ions released from terrestrial POM in seawa-ter, can thus strongly impact on the preservation of terrestrial POM.

ort Sea during summer (it is important to note that the arrow sizes indicating thegradation, could differ depending on the OM component considered).

J.-F. Rontani et al. / Organic Geochemistry 75 (2014) 61–73 71

4.5. Degradation of particulate and sedimentary OM in the Arcticduring summer

To summarize the results obtained here and to link them to pre-vious observations obtained from sinking particles (Yunker et al.,1995; Rontani et al., 2012b) and surface sediments (Yunker et al.,1995, 2005; Goñi et al., 2000, 2005; Stein and Macdonald, 2004;Drenzek et al., 2007; Stein, 2008; Rontani et al., 2012a) from thesame area, we propose a scheme summarizing the degradation ofterrestrial and marine POM in the Mackenzie Shelf during summer(Fig. 7). Initially, the Mackenzie River discharges a high amount ofterrigenous OM to the shelf. Goñi et al. (2005) estimated that onlyca. 4% of the total OC input (both terrigenous and marine) is buriedin the Beaufort Shelf. On the basis of the results of the present ste-rol degradation study, intense biotic and abiotic degradation ofvascular plant material in SPM seems to intervene, contributingto the disappearance of terrigenous OM on the shelf. This degrada-tion, involving intense autoxidation and biodegradation, takesplace mainly in the less dense higher plant debris remaining in sus-pension, particularly during advection to sites further offshore. Asmall part of this highly degraded debris reaches the sedimentsafter aggregation (Rontani et al., 2012a). In contrast, terrigenousOM associated with lithic material settles quickly and is onlyweakly degraded before incorporation into sediments (Yunkeret al., 1995). Sinking particles composed mainly of phyto- and zoo-planktonic material undergo intensive photodegradation in theeuphotic zone in summer (Rontani et al., 2012b). These photo-chemical processes also affect bacteria attached to the particles.Due to this photochemical effect on bacteria, biodegradation isnot a dominant process during settling although autoxidation isstill active. This highly abiotically degraded material (sinking par-ticles + aggregated SPM), containing a high amount of harmfulhydroperoxides, induces strong oxidative stress in sedimentarybacteria (presence of unusually high proportions of vaccenic acidwith a trans double bond; Rontani et al., 2012a), limiting bacterialdegradation in surface sediments. This abiotic limitation of biodeg-radation can explain the relative stability of terrigenous materialpreviously observed in surface sediments all across the MackenzieShelf and into the Canada Basin (Yunker et al., 1995; Belicka et al.,2004). In deeper sediments, this stress disappears progressivelyand planktonic material is quickly degraded while terrestrial com-ponents appear to be more preserved.

5. Conclusions

The possibility of enhanced release of C from terrestrial soils dueto permafrost degradation has great potential to alter the input ofOC to the Beaufort Sea (Goñi et al., 2005). Moreover, changes in tun-dra vegetation toward leaf-bearing plants could be induced rapidlyunder the effects of global warming, thereby enhancing the deliveryof modern vascular plant OC to Arctic rivers and, eventually, the Arc-tic Ocean. To estimate the consequences of climate change for thisstrategic zone, a better understanding of the processes controllingdegradation and burial of terrigenous OM is essential. Very highproportions (relative to the parent sitosterol) of 24-ethyl-5a(H)-cholestan-3b-ol and 24-ethylcholestan-3b,5a,6b-triol, resultingfrom bacterial degradation and autoxidation of sitosterol, respec-tively, were detected, particularly in offshore SPM samples fromthe Mackenzie Shelf. These results allowed us to demonstrate that,in surface waters of the Beaufort Sea, biodegradation and especiallyautoxidation strongly affect vascular plant lipids and probably alsothe other components of terrigenous OM delivered by the Macken-zie River. Despite this extensive degradation in surface waters, asmall fraction of the terrigenous OM clearly survives and is incorpo-rated into the sediments (Yunker et al., 1995; Belicka et al., 2004). In

this zone, the assumption that prior degradation of terrigenous OMduring transit to the sea should result in good preservation of theremaining material within the marine environment is clearly notappropriate.

Acknowledgements

The study was conducted as part of the MALINA ScientificProgram funded by ANR (Agence Nationale pour la Recherche)and French and European Space Agencies. We would like to thankM. Babin, chief scientist for the cruise and coordinator of the MALI-NA program. We are very grateful to the crew of the CanadianIcebreaker CCGS Amundsen for assistance at sea. Special thanksare due to J. Para for help during the cruise and to C. Marec andY. Graton for water sampling. The 2011 Mackenzie River samplingwas part of the Arctic Great Rivers Observatory (Arctic-GRO,US-NSF # 0732522) and a Rubicon postdoctoral grant for J.E.V(NL-NWO # 825.10.022). This work was carried out within theframework of the transverse axis DEBAT (DEgradation Biotique etAbiotique de la maTière organique en milieu marin: processus etinteractions) of the MIO. Thanks are due to S. Belt and an anony-mous reviewer for helpful and constructive comments.

Associate Editor—M. B. Yunker

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