Molecular and isotopic characterization of the particulate organic matter from an eutrophic coastal...

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Molecular and isotopic characterization of the particulate organic matterfrom an eutrophic coastal bay in SE Brazil

Francine A. Kalas a, Renato S. Carreira b,�, Stephen A. Macko c, Angela L. R. Wagener a

a LABMAM/Department of Chemistry, Pontifical Catholic University, Rio de Janeiro, RJ 20453-900, Brazilb LAGOM/School of Oceanography, Rio de Janeiro State University, Rio de Janeiro, RJ 20550-013, Brazilc Department of Environmental Sciences, Universisty of Virginia, Charlottesville, VA 22903, USA

a r t i c l e i n f o

Article history:

Received 12 January 2009

Received in revised form

31 July 2009

Accepted 4 September 2009Available online 15 September 2009

Keywords:

Molecular markers

Isotopes

Particulate organic matter

Guanabara Bay

a b s t r a c t

The present work aimed at studying the origin of particulate organic matter in Guanabara Bay and in

some rivers of the Guanabara basin by using elemental composition, isotopic ratios (d13C and d15N) and

molecular markers (sterols) in samples collected in two periods (winter and summer). Elemental and

isotopic compositions were determined by dry combustion and mass spectrometry, respectively, while

sterols were investigated by GC–FID and GC–MS. Higher sterol concentrations were present in the

north-western part of the bay in winter (5.10–23.5mg L–1). The high abundance of algal sterols

(26–57% of total sterols), the elemental composition (C/N=6–8) and the isotopic signatures

(d13C=�21.3% to �15.1% and d15N= +7.3% to +11.1%) suggested the predominance of autochthonous

organic matter, as expected for an eutrophic bay, although seasonal variation in phytoplankton activity

was observed. Coprostanol concentration (fecal sterol) was at least one order of magnitude higher in the

particulate material from fluvial samples (4.65–55.98mg L–1) than in the bay waters (o0.33mg L–1). This

could be ascribed to a combination of factors including efficient particle removal to sediments in the

estuarine transition zone, dilution with bay water and bacterial degradation during particle transport in

the water column.

& 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Coastal systems, like estuaries and deltas, play an importantrole in the transport of organic matter from continents to theocean (Hedges and Keil, 1995; Bianchi and Canuel, 2001). Thisderives from the capacity to ‘filter’ the original river input ofparticulate (and dissolved) organic matter both in qualitative andquantitative ways (Schlesinger, 1997; Hobbie, 2000; McCallisteret al., 2006).

There are still major questions regarding the type and amount oforganic matter which is effectively exported and finally accumulatesin coastal and shelf sediments. This is in great part due to differentways by which estuaries operate (Hobbie, 2000; Middelburg andHerman, 2007) and to the presence of allochthonous as well asautochthonous organic matter with distinct reactivity (Hedges et al.,1997; Hedges and Keil, 1999). The burning of fossil fuel, the release ofnutrients (sewage and fertilizer) and the changes in soil use increasethe imbalance between natural/anthropogenic carbon productionand respiration in the coastal zone (Ver et al., 1999; Mackenzie et al.,2002).

Understanding the biogeochemical cycle of organic carbon incoastal systems and the role of these areas in a global scale, involvesknowledge of relative contributions of terrestrial (including theanthropogenic contribution), riverine and marine sources to the poolof organic matter in the water column (dissolved and/or particulated)and in sediments. Furthermore, pre- and post-depositional chemicalchanges (or destruction) of organic matter induced by microbialaction must be accounted for to avoid underestimating fluxes ofselected fractions of organic matter (Hedges et al., 1997).

The very complex task of unraveling the geochemistry oforganic matter in the ocean, especially in the coastal zone,requires information about the isotopic (d13C and d15N) andmolecular composition of organic matter (Saliot et al., 2002;McCallister et al., 2006; Countway et al., 2007; Volkman et al.,2008). Lipids represent a small percentage of particulate organiccarbon in water, but are extensively used as molecular markersdue to their source specificity and resistance to alterationcompared to other forms of organic matter (Saliot et al., 1991;Meyers, 2003; Volkman, 2006).

We designed a core project in Guanabara Bay, Brazil (Fig. 1), aimedat studying the alterations in organic carbon cycling derived fromurban settlement in the drainage basin which started in the 19thcentury and was accelerated during the last 40–50 years. The input ofnutrients associated with raw sewage (Kjerfve et al., 1997; Feema,

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Continental Shelf Research

0278-4343/$ - see front matter & 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.csr.2009.09.007

� Corresponding author. Tel.: +55 21 25877842.

E-mail address: [email protected] (R.S. Carreira).

Continental Shelf Research 29 (2009) 2293–2302

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1998), high rates of primary production (0.17 mol C m�2 year�1;Rebello et al., 1988) and elevated sedimentation rates influenceorganic carbon accumulation and preservation in bay sediments.This was confirmed in a previous work (Carreira et al., 2002), wherewe found a ten-fold increase in carbon accumulation in sediments(4.2–4.7 mol C m�2 year�1), mainly derived from autochthonousprimary production induced by the eutrophic condition in thebay.

In the present study, we determined for the first time inGuanabara Bay the isotopic and lipid (sterol) composition ofsuspended organic particles sampled in the bay and in some of theaffluent rivers. The objective of this work was to evaluatespace–time (winter/summer) variations in allochthonous and auto-chthonous contributions to the organic particulate pool in the bay.

2. Material and methods

2.1. Study site

Guanabara Bay (Fig. 1) lies in the heart of Rio de Janeiro city(221400–231000S and 431000–431180W). The bay has a total area of

384 km2 and mean water depth of 7.7 m (Feema, 1998). Theclimate in the region is tropical humid, with a rainy summer and adry winter; the mean annual temperature and rainfall are 23.7 1Cand 1500 mm, respectively, (JICA, 1994). The hydrographic basin ofGuanabara Bay extends over 4080 km2 and is formed by 45 rivers,although only 6 are responsible for more than 85% of the100759 m3 s�1 mean annual total freshwater runoff. Deforesta-tion for urban settlement especially in the west, north-west andeast sectors of the bay, followed by agriculture activities, is themain cause of soil use changes in the drainage basin.

The complex water circulation pattern is defined by gravita-tional and tidal effects although the prevailing winds may beimportant at specific times. The water exchange between the bayand the South Atlantic is in the order of 16,000 m3 s�1, favored bya combination of factors like residual tidal flow, Coriolis accel-eration, systematic wind effects and frictional effects due to localbathymetry and shore configuration (Kjerfve et al., 1997).

The main topographical feature in Guanabara Bay is a centralchannel with depths of 30–40 m that extends from the mouth tothe inner parts of the bay (Fig. 1). Through this channel, sea waterenters the bay driven by tidal forces which guarantee a relativelyshort mean half-water volume renewal (11.4 days), although in

Fig. 1. Water sampling stations in Guanabara Bay (P1–P6) and rivers (S.J. Meriti, Sarapuı, Iguac-u, Suruı and Guapimirim). Urbanized areas are in dark-gray. The 5m-isobath

is indicated by a dashed line.

F.A. Kalas et al. / Continental Shelf Research 29 (2009) 2293–23022294


some parts of the bay this time is considerably higher (Kjerfveet al., 1997 and references therein).

Salinities and temperatures profiles show a well-mixed watercondition at the entrance of the bay extending 15–20 km inwards.Thereafter, the system is moderately stratified.

Guanabara Bay is one of the most studied coastal systems inBrazil. Individual studies and monitoring programs conducted inthe bay showed the increasing contamination of water andsediments by toxic metals, hydrocarbons, nutrients and patho-genic organisms, as well as changes in pelagic and benthiccommunities since the middle of the last century (Valentinet al., 1999a; Carreira et al., 2004; Fernandez et al., 2005; Netoet al., 2006; Da Silva et al., 2007; Farias et al., 2007; Cordeiro et al.,2008; Souza et al., 2008).

Environmental problems are associated with urban settlementand industrial activities. Domestic effluents derived from about 7million inhabitants are discharged directly into the bay—thelatest available estimates of total organic load resulting fromdomestic raw sewage amounts to 410 ton day�1 (only 15% of thetotal organic load undergo any kind of pre-treatment before finaldisposal). Additional sources of contaminants include effluentsfrom 14,000 industries (the second largest industrial complex inBrazil), two refineries, two commercial harbors, fourteen oilterminals, thirty-two dockyards, marinas, and yacht clubs (Feema,1998). Due to the concentration of major contaminant sources andto less effective water circulation patterns, the western, north-western and eastern sectors of the bay show the poorestenvironmental conditions (Fig. 1).

2.2. Sampling

Samples were collected in July 1999 (winter) and January 2000(summer) in five stations distributed along the north-south axis ofGuanabara Bay (Fig. 1). Water samples were collected at 1 and 5 mwater depths, as well as near the bottom in stations 1 and 2. Forsterol analysis, 3 L of water were pumped at a rate of 200 mLmin�1 by an in situ submerged filtering system (InfiltrexTM100).Suspended particulate matter was retained in two sandwichedpre-ignited (450 1C overnight) GF/F filters (142 mm, 0.7mm). Uponrecovery, the filters were folded, wrapped in pre-ignited (450 1Covernight) aluminum bags and kept refrigerated until arriving tothe laboratory. Additional water collected with the aid of aGo-FloTM bottle was for physical–chemical measurements (sali-nity by the conductometric method and dissolved oxygen byWinkler—Grasshoff et al., 1983) and for determination ofsuspended particulate matter (SPM), particulate organic carbon,total nitrogen (POC and TN) and isotopic (d13C and d15N)composition of particulate organic matter after filtration throughpre-ignited GF/F filters (25 mm; 0.7mm). All filters were stored at�20 1C until analysis.

In the summer of 2000, water from some rivers in theGuanabara Bay watershed (Fig. 1) was sampled by immersingsealed glass bottles (5 L) thereafter opened at �1 m below surface.In the laboratory filtration (0.1 L) was performed in large(142 mm) GF/F filters for sterol determination and in small(25 mm) GF/F filters for elemental analysis and isotopic determi-nations. Filters were stored at �20 1C until analysis.

2.3. Methods

2.3.1. Elemental and isotopic analyses

Filters were thawed and dried at 60 1C to constant weight, andthe total suspended matter (TSM) content was determinedgravimetrically (70.01 mg). Particulate organic carbon (POC)and total nitrogen (TN) were measured in a Carlo Erba Model

EA1110 CHNS analyzer, after removal of inorganic carbon by acidtreatment (vapor of HCl for 24 h—Hedges and Stern, 1984). Tworound plugs (around 2 cm2 each) were sub-sampled from eachfilter and analyzed independently. The final result represents themean of these two analyses. Quantification was performed byresponse factor relative to the standard cystine. Precision of POCand PON analyses was better than 10%.

Filters for isotopic analysis were acidified in the same way asabove described and thereafter dried. The carbonate-free residueswere converted to CO2 and N2 in an elemental analyzer(Carlo Erba EA), which is coupled to an OPTIMA stable isotoperatio mass spectrometer (Micromass, Manchester, UK). Theformed gases are directly injected into the source of the massspectrometer. The stable isotopes are expressed as dNE=[Rsample/Rstandard–1] �103 (%), where N is the heavy isotope of theelement E and R is the abundance ratio of the heavy to lightisotopes (13C/12C, 15N/14N) of that element. The Pee Dee Belemnitelimestone (PDB) and atmospheric N2 (air) were used as thestandards for carbon and nitrogen, respectively. The reproduci-bility of the measurement was typically better than 70.2%.

2.3.2. Sterols extraction and quantification

Filters (GF/F; 142 mm) were freeze-dried and spiked with 5a-androstan-3b-ol as internal standard. Particulate lipids wereSoxhlet extracted for 24 h using dichloromethane. The bulkextract was concentrated in vacuum and transferred to the topof a glass column filled with 2.5 g of silica gel (G-60, Merck,230–400 mesh, 5% deactivated) and 4.6 g of alumina (neutral,5% deactivated). Three fractions were isolated from the bulkextract: 10 mL of n-hexane (aliphatic hydrocarbons), 20 mL ofn-hexane:dichloromethane (1:1 v/v; aromatic hydrocarbons) and20 mL of dichloromethane:methanol (9:1 v/v; sterols and morepolar compounds). Only the sterol fraction will be considered inthis paper. The third fraction was concentrated by vacuumevaporation and a gentle stream of purified N2 and stored at�20 1C until analysis by GC/FID and GC/MS.

Particulate sterols were analyzed using an HP 6890 gaschromatograph fitted with a splitless injector and a flameionization detector (FID). An HP-5MS fused silica capillary columnof 30 m�0.25 mm i.d. (0.25mm film thickness) was used. Thecarrier gas (Helium) was maintained at a constant flow rate of1 mL min�1, and nitrogen was used as the make-up gas. Thetemperature program in the oven was 60 1C initial, from 60 to300 1C at 6 1C min�1 and a final hold of 29.5 min. Before injection,sterols were derivatized to their trimethylsilyl (TMS) derivativeswith bis(trimethylsilyl)trifluoroacetamide (BSTFA) at 80 1C for 1 h.Peaks were identified based on retention times of authenticstandards (5b-cholestan-3b-ol, cholest-5-en-3b-ol, 5a-cholestan-3b-ol, 24-methylcholest-5-en-3b-ol, 24-ethylcholesta-5,22-dien-3b-ol and 24-ethylcholest-5-en-3b-ol). Confirmatory analyses, aswell as the identification of additional sterols, were performed byGC/MS on selected samples. Samples were run in a Thermo-Finnigan Trace GCQ system operating in the EI mode (70 eV)and at full scan mode (m/z 50–500). Compounds were separatedusing an HP5-MS low bleed fused silica capillary column(30 m�0.25 mm i.d�0.25mm film thickness). The oven tempera-ture program used in the GC/MS system was similar to that usedin the GC/FID.

Sterols were quantified based on the GC/FID areas and on theresponse factors of standards relative to 5a-androstan-3b-ol. Forthose sterols identified only by GC/MS, quantification was basedon relative response factors of sterols with similar chemicalstructures. Throughout the study the instrument performance(linear response and relative response factors) was regularlychecked by injection of standard solutions of all sterols

F.A. Kalas et al. / Continental Shelf Research 29 (2009) 2293–2302 2295


(ranging from 50 to 175 ngmL�1). Procedural blanks showed nocontamination. Detection limits ranged from 0.01 to 0.03mg L�1

for all sterols. Data were corrected for recovery of the internalstandard (mean of 93.8728.8% for n=23).

3. Results and discussion

3.1. Water column characteristics

Salinity varied over a narrow range considering the twosamplings (Table 1), with a mean value of 30.671.4. There wasno clear horizontal gradient in salinity along the south/north axisof the bay, indicating an efficient water mixing induced by tidalcurrents. There was also no significant (po0.01) differences insalinity between the two samplings. However, a slightstratification in the water column was observed, since highersalinities (up to 32.9 at station 6) were observed in waters close tothe bottom.

Contrary to the salinity distribution, other parameters showedseasonal variability. The mean concentration of total suspendedparticulate matter (SPM) was lower in winter (�30 mg L�1) thanin summer (�50 mg L�1), with the highest variation observed atstations 3 and 4 (Table 1). In general, higher values of SPM wereobserved in the upper estuary (stations 3–6) (20.0–58.5 mg L�1 inwinter and 43.0–65.2 mg L�1 in summer) while lower values weremeasured in summer (25.4–47.4 mg L�1) at stations 1 and 2, in thesouthern part of the bay (Table 1). In the same way, dissolvedoxygen supersaturation was present in the winter samples(146741%) throughout the water column, while lower levelswere measured in the summer samples (81725%), especially inthe bottom samples at stations 4 and 6 (O2 saturation=45.9% and49.7%; Table 1).

3.2. Elemental and isotopic composition of particulate organic

matter

The distribution of particulate organic carbon was character-ized by elevated concentrations for samples collected at the

surface and in the northern region of the bay (stations 4–6;Table 1). Particularly carbon-enriched particles were collected inwinter (POC=9.94–17.43 mg L�1 or 10–40% SPM) in comparison tosummer samples (POC=3.8–6.2 mg L�1 or 5–10% SPM). In bothsamplings these values were higher than measured in thesouthern bay, as can be seen by our data at station 1(POC=2.67–3.19 mg L�1) and other values published recently forthe same region (POC=1.40–1.68 mg L�1 Guenther et al., 2008).Particulate nitrogen (PN; Table 1) was closely related to POC andthe regression line relating the two variables crosses the axis veryclose to the origin (PN=0.22+0.13POC), indicating the predomi-nance of organic nitrogen in the suspended particles.

The overall mean C/N molar ratio of 6.771.3 (n=19; winterand summer samples) was in the range usually ascribed to marinephytoplankton (Meyers, 1997; Maksymowska et al., 2000; Quigget al., 2003) and thus suggests major contribution of autochtho-nous sources to the pool of suspended organic matter in the bay.This is consistent with the net autotrophic metabolism (i.e., ratesof primary production higher than bacterial respiration) measuredin the bay’s surface waters (Guenther et al., 2008). On the otherhand, samples 3s, 3m, 4m and 6s collected in summer show lowC/N (o 5.0) and dissolved oxygen saturation ranging from 45.8%to 84.0% (Table 1), which indicated the presence of degradedparticulate organic matter.

The carbon isotopic composition of POC showed distinct values(po0.001; T-test) between the two samplings (Table 1). In winter, thed13C ranged from �15.2% to �18.9% (mean7sd=�16.971.3%)with particularly enriched values for the surface samples, while in thesummer samples the d13C values (�18.6% to �21.3%, mean7sd=�19.870.8%) were in the range usually found in estuariesdominated by autochthonous organic matter (Sigleo and Macko,2002; Liu et al., 2007). In the warm (mean annual temperatures of25.272.5 1C at the surface) and eutrophic waters of Guanabara Bay(Kjerfve et al., 1997), the rate of primary production is limited by lightand thus are influenced by short-term variation in environmentalconditions (e.g., tidal oscillation and continental runoff) rather thanseasonal factors (Valentin et al., 1999b; Guenther et al., 2008). In thissense, the higher concentration of dissolved oxygen (as well aselevated % saturation levels; Table 1) in winter suggest enhanced

Table 1Salinity, dissolved oxygen (O2dis; mg L�1 and % of saturation), suspended particulate matter (SPM), particulate organic carbon (POC; mg L�1 and % of SPM), particulate

nitrogen (PN), C/N molar ratio and carbon and nitrogen isotopic composition of particulate organic matter in the two samplings in Guanabara Bay.

Stations Sampling

depth (m)

Local

depth

(m)

Secchi

depth

(m)

Water

temp.

(1C)

Salinity O2dis

(mg L�1)

O2dis

(% sat.)

SPM

(mg L�1)

POC

(mg L�1)

POC

(%TPM)

PN

(mg L�1)

C/N

(molar)d13C

(%)

d15N

(%)

Winter

1 o1 29 1.3 23.4 30.7 12.3 173.0 25.28 2.77 11.0 0.54 5.94 �18.6 8.9

5 23.0 31.2 8.2 115.0 34.32 2.67 7.8 0.56 5.58 �18.6 8.1

15 22.1 – 8.0 – 27.80 3.19 11.5 0.62 5.98 �18.9 8.0

2 o1 12 1.0 25.0 30.1 13.9 199.0 25.56 11.42 44.7 1.79 7.44 �16.6 10.2

5 23.0 30.8 7.1 172.0 25.38 4.06 16.0 0.86 5.54 �17.9 9.9

10 23.5 31.5 4.3 116.0 47.44 4.46 9.4 0.82 6.35 �16.6 11.1

3 o1 12 1.5 26.0 29.4 11.9 116.0 20.67 3.19 15.4 0.61 6.10 �16.2 10.6

5 25.0 – 8.6 – 20.70 4.09 19.7 0.83 5.74 �16.3 9.7

4 o1 8 1.0 25.0 28.7 11.5 164.0 35.25 9.94 28.2 1.62 7.15 �15.2 9.3

5 25.0 29.4 7.0 100.0 40.67 2.59 6.4 0.48 6.31 �15.8 9.5

5 o1 6 1.0 27.0 32.3 13.8 207.0 35.62 10.66 29.9 1.52 8.17 �15.1 9.3

6 o1 8 1.0 27.5 – 14.7 – 58.50 17.43 29.8 2.56 7.94 �15.8 9.6

5 23.5 32.9 7.2 99.0 50.43 4.07 8.1 0.86 5.51 �17.8 8.8

Summer

3 0o1 12 0.5 27.7 30.6 5.6 84.0 63.60 3.73 5.9 0.88 5.10 �19.3 7.8

5 24.6 31.5 5.4 77.4 52.30 2.77 5.3 0.67 4.83 �19.9 7.3

4 o1 8 0.5 25.0 27.4 7.1 99.9 65.20 3.79 5.8 0.77 5.75 �20.2 7.5

5 23.3 31.5 3.3 45.8 52.00 2.70 5.2 0.68 4.62 �19.9 8.0

5 o1 6 0.5 27.3 29.1 7.1 106.0 43.00 6.21 14.4 1.25 5.81 �21.3 8.1

6 o1 8 0.5 26.8 30.7 7.0 104.0 48.90 4.09 8.4 0.79 6.03 �19.8 8.1

5 25.3 32.3 3.4 49.7 48.60 1.30 2.3 – – �18.6 8.0



phytoplankton activity and thus incorporation of 13C enriched HCO3�.

This process was probably favored by increased light penetration inthe water column during winter, as shown by Secchi depths(winter=1.170.2 m and summer=0.5 m; Table 1). The relativelydepleted carbon signature observed in summer could be derivedfrom larger terrestrial inputs observed in the rainy season.

The mean d15N was 9.570.9% in winter and 7.870.3% insummer (Table 1). Overall, these values suggest phytoplanktonassimilation of N derived from remineralized organic matter inGuanabara Bay, as observed in other estuarine systems (Middel-burg and Nieuwenhuize, 2001; Sigleo and Macko, 2002;Pinon-Gimate et al., 2009). It is noteworthy that winter sampleshave extremely enriched values for both d15N and d13C (Fig. 2),which suggests that during high phytoplankton activity (seeprevious discussion based on d13C) there is incorporation ofisotopically heavy residual NH4

+, since this is the dominantmechanism for 15N enrichment in particulate organic matter inestuaries (Liu et al., 2007).

3.3. Composition and distribution of particulate sterols

Sixteen sterols were identified in the suspended particulatematter collected in Guanabara Bay and in some rivers from thewatershed (Tables 2 and 3). The concentrations of total sterols inthe winter ranged from 1.65 to 23.8mg L�1, with higher valuesappearing in the northern region (stations 4–6) at the surface and/or at 5 m water depth (Table 2). A similar distribution of totalsterols was observed in summer, although concentrations wererelatively lower (0.85–15.05mg L�1; Table 3).

Total particulate sterol concentrations in Guanabara Bay weregenerally higher than reported for coastal systems from temperateand/or sub-tropical regions, like the Krka River estuary in theAdriatic Sea (0.44–0.72mg L�1—Laureillard and Saliot, 1993), theChang Jiang River estuary in China (0.06–5.91mg L�1—Sicre et al.,1994), and in the USA, the Delaware estuary (�1.0–6.0mg L�1;Mannino and Harvey, 1999), Florida Bay (0.09–3.04mg L�1; Xu andJaffe, 2007) and Chesapeake Bay (1.0–8.0mg L�1; Canuel andZimmerman, 1999).

The TOC-normalized concentration of total sterols (Table S1,supplementary material) showed an opposite trend in relation tothe distribution of absolute values (in mg L�1), as discussed above.First, excluding station 3, the normalized concentration of sterolswere higher in summer compared to winter (stations 1 and 2 werenot sampled in summer). In addition, maximal concentrations oftotal sterols were measured in winter at the surface and 5 msamples from station 1 (3.370.3mg mg Corg

�1), located in thesouthern part of the bay, while at stations 4–6 (which showedhigher values in mg L�1) the normalized total sterols concentra-tions were relatively lower (1.5–2.0mg mg Corg

�1) (Fig. 3).The nature of particulate organic matter can be assessed by

grouping individual sterols derived from autochthonous (plankton-derived) or allochthonous (vascular plant-derived) sources (Canueland Zimmerman, 1999; Countway et al., 2007). In the present work,we considered as autochthonous sterols the following compounds:24-norcholesta-5,22-dien-3b-ol, 27-nor-24-methylcholesta-5,22-dien-3b-ol, cholesta-5,22-dien-3b-ol, cholest-5-en-3b-ol, 24-methyl-cholesta-5,22-dien-3b-ol, 24-methylcholesta-5,24(28)-dien-3b-ol and4a,23,24-trimethylcholest-22-en-3b-ol. The allochthonous sterolsinclude 24-methylcholest-5-en-3b-ol, 24-ethylcholesta-5,22-dien-

Fig. 2. Plot of d13C versus d15N of SPM from Guanabara Bay. s: surface. m: mid

water, b: bottom.

Table 2Sterols in suspended particulate matter from Guanabara Bay in winter (June 1999).

Sterols Stations in Guanabara Bay

1 2 3 4 5 6

Surface 5 m 15 m Surface 5 m 10 m Surface 5 m Surface 5 m Surface Surface 5 m

24-nor-cholesta-5,22(E)-dien-3b-ol 0.11 0.18 0.02 0.19 0.11 0.13 0.31 0.29 0.41 0.30 0.56 0.31 0.32

27-nor-24-methylcholest-5,22-dien-3b-ol 0.03 0.03 0.02 0.08 0.02 0.02 0.20 0.06 0.25 0.17 0.18 0.81 0.11

5b-cholestan-3b-ol 0.16 0.33 0.05 0.09 0.03 0.11 0.01 nd 0.04 0.01 0.04 0.08 nd

5b-cholestan-3a-ol 0.07 0.02 0.02 nd 0.01 0.06 0.03 nd nd 0.01 0.08 0.10 nd

cholesta-5,22-dien-3b-ol 0.26 0.47 0.11 0.49 0.23 0.33 1.62 0.80 1.57 1.16 1.45 2.52 0.75

5a-cholestan-22-en-3b-ol 2.29 0.28 0.04 0.22 0.16 0.07 0.62 0.68 0.20 0.18 0.13 3.84 1.21

cholest-5-en-3b-ol 1.33 1.89 0.35 1.47 0.81 0.98 0.65 0.76 1.30 0.91 1.55 1.86 0.73

5a-cholestan-3b-ol 0.12 0.26 0.04 0.17 0.07 0.21 0.09 0.14 0.20 0.18 0.41 0.45 0.15

24-methylcholesta-5,24(28)-dien-3b-ol 2.00 3.23 0.21 2.73 1.37 1.20 0.86 0.37 nd 0.81 nd 0.17 0.46

24-methylcholesta-5,22-dien-3b-ol 0.38 0.83 0.16 0.98 0.37 0.09 0.78 0.57 1.26 0.75 0.72 1.80 0.51

24-methylcholest-5-en-3b-ol 0.24 0.37 0.01 0.02 0.14 0.28 0.35 0.08 3.16 0.21 2.64 2.53 0.10

24-methyl-5a-cholestan-3b-ol 0.45 0.02 0.02 nd 0.07 0.53 0.10 nd 0.35 0.36 0.20 0.33 0.13

24-ethylcholest-5,22-dien-3b-ol 0.16 0.39 0.08 0.31 0.14 0.25 0.32 0.19 0.87 0.24 0.34 1.98 0.20

24-ethylcholest-5-en-3b-ol 0.58 0.77 0.24 0.93 0.50 0.69 0.29 0.94 0.74 0.96 0.60 2.37 0.77

24-ethyl-5a-cholestan-3b-ol 0.06 nd 0.02 0.01 nd 0.01 0.59 nd 0.87 0.15 nd 0.47 0.09

4a,23,24-trimethylcholest-22-en-3b-ol 0.13 0.47 0.10 0.54 0.07 0.16 0.97 0.22 3.71 0.11 3.97 3.91 0.13

Total sterols 8.37 9.54 1.49 8.23 4.10 5.12 7.79 5.10 14.9 6.51 12.9 23.5 5.66

Values in mg L�1 (nd=not detected oro0.01mg L�1).



3b-ol and 24-ethylcholest-5-en-3b-ol. The result (Fig. 4) showspredominance of autochthonous sources of sterols in bothsamplings consistent with the eutrophic conditions in the bayas also indicated by the elevated density of phytoplankton(107–108 cells L�1 for each group of diatoms, dinoflagellates andcyanobacteria in the summer; Valentin et al., 1999b) and by rates ofnet primary production of 0.17 mol C m�2 day�1 (Rebello et al., 1988).

The general indication of autochthonous and allochthonoussources of particulate organic matter can be traced in furtherdetail through the relative contribution of individual compoundsto total sterols concentrations (Table S2, supplementary material).In the winter sampling, five sterols made up about 60% of totalsterol content, however there were significant differences in therelative contribution of each compound in different stations.In the southern portion of the bay (stations 1 and 2),24-methylcholesta-5,24(28)-dien-3b-ol comprised 23.9–33.9% oftotal sterols, followed by cholest-5-en-3b-ol (cholesterol; 15.9–21.2%). The sterol 24-methylcholesta-5,24(28)-dien-3b-ol ismainly produced by diatoms, while cholesterol is abundant inzooplankton and in some species of phytoplankton (Volkman,1986). Recent report indicated that Skeletonema spp., enriched in24-methylcholesta-5,24(28)-dien-3b-ol (Volkman et al., 1998),was relatively more abundant from June to October (dry period) inthe outer portion of Guanabara Bay in comparison to the wetperiod (Santos et al., 2007). The composition of sterols in POMsuggests the occurrence of a diatom bloom at the entrance of thebay, possibly more intense at station 2 due to the higher POCconcentration (11.42 mg L�1; Table 2). The 24-methylcholesta-5,22-dien-3b-ol, which may be related to diatoms but is alsoproduced by haptophytes and cryptophytes (Volkman, 2006 andreferences there in) was less abundant and represented only8–10% of total sterols.

Still in relation to the winter samples, in the northern part ofthe bay (stations 4–6) the sterol composition was different fromthat in the southern portion (stations 1 and 2). High contributionof 4a,23,24-trimethylcholest-22-en-3b-ol (dinosterol; 16.4–30.9%of total sterols; Supplementary material) was observed, while theabundance of 24-methylcholesta-5,24(28)-dien-3b-ol decreasedto 8.1–12.4% (Supplementary material). The high levels of

dinosterol, a specific marker for dinoflagellates (Robinson et al.,1984; Volkman et al., 1998), at stations 4–6 in winter (and also insummer) sampling points out the predominance of dinoflagellatesover diatoms in the SPM. This is consistent with the generalcharacteristics of plankton distribution in the bay: high densities

Table 3Sterols in suspended particulate matter from Guanabara Bay and in selected rivers from the watershed in summer (January 2000).

Sterols Stations in Guanabara Bay Riversa

3 4 5 6

Surface 5 m Surface 5 m Surface Surface 5 m Iguac-u Sarapuı S.J. Meriti Suruı Guapi

24-nor-cholesta-5,22(E)-dien-3b-ol nd 0.15 0.29 0.32 0.26 0.29 0.10 nd nd nd nd 1.15

27-nor-24-methylcholesta-5,22-dien-3b-ol nd 0.02 0.12 0.09 0.21 0.14 0.02 nd nd nd 0.19 nd

5b-cholestan-3b-ol nd 0.02 0.05 0.01 nd 0.03 0.04 12.99 25.45 55.98 4.65 5.33

5b-cholestan-3a-ol nd 0.02 0.02 0.01 nd 0.08 0.05 4.10 7.91 9.09 0.14 nd

cholesta-5,22-dien-3b-ol 0.08 0.13 1.03 0.82 1.21 1.01 0.13 nd nd nd 0.42 nd

5a-cholestan-22-en-3b-ol nd 0.06 0.08 0.10 0.11 0.11 nd 0.34 nd nd 1.02 nd

cholest-5-en-3b-ol 0.22 0.24 1.52 1.04 2.84 2.02 0.09 16.08 24.52 24.39 0.53 1.01

5a-cholestan-3b-ol 0.02 0.08 0.08 0.14 0.24 0.39 0.11 3.47 5.30 7.69 0.59 0.25

24-methylcholesta-5,24(28)-dien-3b-ol 0.12 0.10 0.60 0.48 1.29 0.85 0.03 3.98 4.82 9.96 1.35 nd

24-methylcholesta-5,22-dien-3b-ol 0.10 0.14 0.90 0.47 1.45 1.62 0.08 nd 2.06 nd nd 1.18

24-methylcholest-5-en-3b-ol 0.04 0.07 0.59 0.28 0.82 0.51 0.03 1.37 0.97 1.61 0.41 0.10

24-methyl-5a-cholestan-3b-ol 0.07 0.02 0.72 0.30 0.12 0.78 0.01 nd nd nd nd 1.03

24-ethylcholesta-5,22-dien-3b-ol 0.09 0.04 0.68 0.09 0.86 0.33 0.04 1.42 2.65 1.32 0.38 0.75

24-ethylcholest-5-en-3b-ol 0.25 0.11 4.24 1.13 3.83 1.51 0.03 3.91 4.65 4.94 0.82 1.04

24-ethyl-5a-cholestan-3b-ol nd 0.03 0.42 0.06 0.60 nd nd 1.04 nd nd nd nd

4a,23,24-trimethylcholest-22-en-3b-ol 0.04 0.08 0.80 0.36 1.21 2.19 0.09 0.20 nd nd nd nd

Total sterols 1.03 1.31 12.1 5.70 15.1 11.9 0.85 48.9 78.3 115 10.5 11.8

Values in mg L�1 (nd=not detected oro0.01mg L�1).

a Samples in rivers collected at the surface.

10.0

1.0

2.0

3.0

4.0

5.0

Tota

l ste

rols

(µg

mg C

org-1

)

winter-1999 summer-2000

2 3 4 5 6

Fig. 3. TOC-normalized concentration of total sterols in winter-1999 and summer-

2000. Columns represent mean concentrations of surface and 5 m depth samples

and whiskers the maxima values.



of diatoms at the entrance of the bay and dinoflagellates (andcyanobacteria) more abundant in the northern part (Valentinet al., 1999b; Santos et al., 2007). The distribution of cholesterol(high percentages in stations 1 and 2) is also consistent with theobservation of a higher density of zooplankton in the southernportion of the bay (Valentin et al., 1999b).

In the summer sampling, the most relevant change in steroldistribution, when compared to winter results, was an increase inabundance of 24-ethylcholest-5-en-3b-ol in the northern part ofthe bay, especially at stations 4 and 5 (25.4–34.9% of total sterols;Table S2, Supplementary material). Unfortunately, there were nodata on stations 1 and 2 in summer for comparison. The origin of24-ethylcholest-5-en-3b-ol in aquatic systems is dubious becauseit is not possible to determine, under ‘usual’ analytical conditions,the isomeric position of the 24-ethyl group (the 24-a isclionasterol, derived from plankton, while the 24-b isomer isb-sitosterol, derived from vascular plants) (Volkman, 2006 andreferences there in). To overcome such limitation, one mayconsider the ratio 24-methylcholest-5-en-3b-ol:24-ethylcholes-ta-5,22-dien-3b-ol:24-ethylcholest-5-en-3b-ol: values between1:1.4:4.0 and 1:1.6:6.0 indicate terrestrial vascular plant as thesource of such compounds, while lower ratios suggest they derivefrom algae (Volkman, 1986; Laureillard and Saliot, 1993). In oursamples collected in summer, the mean value (considering onlythe surface samples) of 1:1.3:5.3 for this ratio indicates that24-ethylcholest-5-en-3b-ol derives from terrestrial/riverineorganic matter. Although this result may be taken as a confirma-tion of the relatively higher contribution of allochthonous sterolsto particulate organic matter at stations 4 and 5 in the summersampling (Fig. 4), the elemental and isotopic compositionpoint out the predominance of autochthonous sources. Such

apparent discrepancy may arise from the influence of cyanobac-teria which may produce 24-ethylcholest-5-en-3b-ol (Volkman,2005) and are abundantly present (density of 105–108 cells mL�1

Valentin et al., 1999b) in the northern portion of the bay duringthe wet (summer) season. In such conditions, the proposed ratiomay be misleading as source indicator.

The low levels of reduced sterols, like 5a-cholestan-3b-ol,24-methyl-5a-cholestan-3b-ol and 29-ethyl-5a-cholestan-3b-ol,typically comprising less than 3–5% of total sterols in bothsamplings, suggests that POM is principally composed of ‘‘fresh’’phytoplankton material (Wakeham and Canuel, 2006). The role ofbacteria in the transformation of POM is not yet well understoodin Guanabara Bay although recent finding of Guenther et al.(2008) emphasizes the effectiveness of auto- and heterotrophic-bacteria in cycling dissolved organic matter (DOM) and POM. Forthis, a specific lipid biomarker must be identified and determined.

Sterols directly associated with sewage material, like5b-cholestan-3b-ol (coprostanol) and 5b-cholestan-3a-ol (epico-prostanol), are present in low concentrations (o0.01 to 0.16mgL�1; Tables 2 and 3) and represent in most cases less than 2% oftotal sterols. There was a significant decrease in the concentrationof coprostanol from river stations (4.6–56mg L�1; see nextdiscussion) to bay sampling sites (o0.3mg L�1), which can beascribed to a combination of factors like (i) efficient removal ofparticles from the water column to sediments in the riverestuarine zone; (ii) dilution of river particles with bay water withlow coprostanol concentration and (iii) degradation of coprostanolby bacterial activity during particle transport. Based on these data,the relative high concentrations of coprostanol found by Carreiraet al. (2004) in bay sediments at station distant from the shoreline(up to 40mg g�1) are possibly derived from transport of sewage

Fig. 4. Distribution of groups of sterols in suspended particulate matter from Guanabara Bay collected at the surface in winter-1999 and summer-2000 (except for stations

1 and 2). Autochthonous (plankton-derived) sterols represents the sum of 24-norcholesta-5,22-dien-3b-ol, 27-nor-24-methylcholesta-5,22-dien-3b-ol, cholesta-5,22-dien-

3b-ol, cholest-5-en-3b-ol, 24-methylcholesta-5,22-dien-3b-ol, 24-methylcholesta-5,24(28)-dien-3b-ol and 4a,23,24-trimethylcholest-22-en-3b-ol. Allochthonous

(vascular plant-derived) sterols include: 24-methylcholest-5-en-3b-ol, 24-ethylcholesta-5,22-dien-3b-ol and 24-ethylcholest-5-en-3b-ol.



particles by deposition/resuspension processes in shallow areasdriven by tidal currents, instead of direct deposition from thewater column.

3.4. Principal component analysis (PCA) of data

A PCA was used to better identify grouping of end members aswell as to distinguish their contribution in each sample.The analysis included the isotopic data, C/N ratio, disso-lved oxygen, sterols which are markers of land biomass (24-methylcholest-5-en-3b-ol, 24-ethylcholesta-5,22-dien-3b-ol and24-ethylcholest-5-en-3b-ol) and sterols from marine biomass(24-methylcholesta-5,24(28)-dien-3b-ol, cholest-5-en-3b-ol, 4a,22,24-trimethylcholest-22-en-3b-ol). Original data were normal-ized dividing each value by the sum of variables in the sample andthen calculating the z-score for each variable. Three componentsexplained 84% of the total variance: d13C (loading of 0.727), d15N(loading of 0.778) the sum of land biomass markers (loading of�0.849), cholesterol (loading of �0.697), and dinosterol (loadingof �0.839) were highly correlated in factor 1; O2dis (loading of�0.821) and C/N (loading of �0.616) are correlated in factor 2and 24-methylcolest-5,24(28),dien-3b-ol (loading of 0.885) is infactor 3. Fig. 5a and b shows the results for the PCA analysis anddetailed data for factor loadings are presented as Supplementarymaterial (Table S3). In Fig. 5a winter samples 4 s, 5 s and 6 s arewell separated from others and seem low in diatoms (see Fig. 5b)and high in dinoflagellates, and possibly zooplankton (highcholesterol that, however, can also derive from sewage) withsome contribution of land derived biomass, despite the enrichedisotope signature. This seems to confirm the hypothesis ofisotopically heavy bicarbonate uptake as carbon source in theinner area of the bay were primary production is known to be veryhigh as demonstrated by the elevated oxygen concentrations (and% saturation level) in the water. The other winter samples show anincreasing contribution of diatoms (see Fig. 5b). These featuresalso confirm the discussions presented above. In the summer,samples 4m, 3s, 3m, and 6m (not shown in the plot) arediscriminated from samples 4s, 5s and 6s because they seem tobear low content of diatoms, dinoflagellates and zooplankton, andcarry C/N ratios r5. These set of observations seem to confirmthe greater influence of bacterial degradation of particulateorganic matter in these samples.

Although the numerous components in the POM makeimpossible the exact estimation of end-member isotope signa-tures by using data for bulk material, the evaluation of the entiredata set obtained in the present work suggests the followinggroups of distinct isotopic signature: (1) samples with degradedparticulate organic matter d15N in the range of 7.670.3% andd13C �19.670.4%; (2) dinoflagellates formed in inner areas ofthe bay d15N in the range of 9.470.2% and d13C �15.470.4%;and (3) diatoms formed in areas near the bay mouth d15N in therange of 9.171.1% and d13C �17.971.1%. Comparison of thesevalues with literature data is difficult first because isotopicinformation for individual algal species is scarce and second butmore important the isotopic signature of the precursors (DIN, DIC,DOC) changes largely depending on local conditions. It should alsobe expected differences within Guanabara Bay dependingwhether the observed site is nearer to sewage sources or isinfluenced by fresh seawater.

3.5. Sterols in the rivers

The suspended particulate matter in the rivers is enriched intotal sterols by at least one order of magnitude in comparison tobay water. This is especially true in Iguac-u, Sarapuı and S.J. Meriti

rivers which are located in the north-western region of the bay(48.9–115mg L�1 of total sterols; Table 3). The most abundantsterol in fluvial POM was coprostanol (26.6–48.7% of total sterols),followed by cholesterol (5.0–32.8% of total sterols). Therefore, theSPM in the studied rivers was composed at the sampling time bysignificant fraction of sewage material. Values of the ratio 5b/(5b+5a)sterol (coprostanol/coprostanol+cholestanol) higher than0.79 found in all rivers are in the range proposed by Grimalt et al.(1990) as indicative of sewage contamination.

Cordeiro et al. (2008) report concentrations of coprostanol inSPM of the Iguac-u River in 2004 which are of the same magnitudeof those obtained in the present work; however coprostanone andepi-cholestanol were present in higher concentrations thancoprostanol. In our samples steroidal ketones were not quantifiedand epi-cholestanol was not detected. The 5a-cholestan-3b-ol(Nishimura, 1982) produced by bacterial activity comprised in oursamples almost 10% of the total sterols (Table 3). The presence inPOM of degradation products of sterols in the present study whichare different from the reported by Cordeiro et al. (2008) suggeststhat bacterial activity in the Iguac-u and probably in the otherrivers of the Guanabara basin is subjected to temporal fluctuationsreflecting distinct environmental conditions at the sampling time(temperature, salinity, etc.).

1s

1m

1b2s

2m

2b

3s3m

4s

4m

5s

6s

6m 3S

3M

4S4M

5S

6S

-2.5FACTOR 1: 41%

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

FAC

TOR

2:

26%

1s

1m

1b2s

2m

2b

3s3m

4s

4m

5s

6s

6m 3S

3M

4S4M

5S

6S

>O2Dis>C/N

>Dinoflag; >Cholest; > Allocto

1s

1m

1b

2s

2m

2b

3s

3m

4s

4m

5s6s

6m

3S3M

4S4M

5S6S

-2.0FACTOR 1: 41%

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5FA

CTO

R 3

: 17%

1s

1m

1b

2s

2m

2b

3s

3m

4s

4m

5s6s

6m

3S3M

4S4M

5S6S

> Diatom

>Dinoflag; >Cholest; > Allocto

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5

Fig. 5. Principal component analysis. Higher case stands for summer samples and

lower case for winter samples. s: surface; m: mid water; b: bottom.



In addition to fecal sterols, sterols grouped as derived fromallochthonous sources (24-methylcholest-5-en-3b-ol, 24-ethyl-cholesta-5,22-dien-3b-ol and 24-ethylcholest-5-en-3b-ol) werealso present in fluvial POM. This was expected since the studiedrivers drain through areas of mangrove forests and C3 plants, butthe low contribution of these sterols in POM of GuanabaraBay indicated that the fluvial contribution of minor significance incomparison to primary productivity in the bay itself. In the morecontaminated rivers (S.J. Meriti, Sarapuı and Iguac-u), a sewagecontribution of 24-ethylcholest-5-en-3b-ol must be accountedfor, since this sterol was found in the raw sewage collected at alocal treatment plant (Cordeiro et al., 2008). Finally, theoccurrence of 24-methylcholesta-5,24(28)-dien-3b-ol in the flu-vial POM (Table 3) indicated the presence of diatom in the rivers(Volkman, 2006). Since autochthonous production in the studiedrivers is severely constrained by low water transparency (Feema,1998; Cordeiro et al., 2008), diatoms in fluvial POM must havebeen originated in the bay and transported to the river by tidalaction.

4. Conclusions

The isotopic and molecular composition of particulate materialin Guanabara Bay showed a strong autochthonous contribution,confirming the elevated euthrophic condition observed in theregion. Enriched carbon isotopic compositions of POC wereobserved in winter and this process was probably driven byenhanced production by dinoflagellates in the northern portion ofthe bay. Diatoms and zooplankton were more abundant in thesouthern part of the bay. Based on the d15N signature, nitrogenavailable for primary production in the both samplings wasderived from remineralized organic material, probably reflectingthe incorporation of NH4

+ derived from sewage input to the bay.The contribution of terrestrial sources was more relevant insummer, possibly reflecting the increased fluvial input during thewet period. However, even under such wet conditions the fluvialcontribution is of minor importance and overwhelmed by theautochthonous production in the bay.

Acknowledgements

Brazilian Research Council (CNPq) for the financial support andfellowship for F.A. Kalas. The comments provided by twoanonymous reviewers enhanced the final version of the manu-script.

Appendix. Supporting Information

Supplementary data associated with this article can be foundin the online version at doi:10.1016/j.csr.2009.09.007.

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