Biogeochemistry of sterols in plankton, settling particles and recent sediments in a cold ocean ecosystem (Trinity Bay, Newfoundland)

Marine Chemistry 76 Ž2001. 253–270 www.elsevier.comrlocatermarchem

Biogeochemistry of sterols in plankton, settling particles and recent sediments in a cold ocean ecosystem žTrinity Bay, Newfoundland/ Edward D. Hudson, Christopher C. Parrish ) , Robert J. Helleur Ocean Sciences Centre and Department of Chemistry, Memorial UniÕersity of Newfoundland, St. John’s, Newfoundland, Canada A1C 5S7 Received 18 September 2000; received in revised form 20 August 2001; accepted 23 August 2001

Abstract In the context of a multidisciplinary study to determine current and past ecosystem health and the relative contributions of sources of organic matter Žmarine vs. terrestrial and natural vs. anthropogenic input., sterols were determined in plankton, settling particles and sediments from Trinity Bay, Newfoundland, a sub-polar Atlantic Ocean ecosystem. The centric diatoms Chaetoceros spp., Thalassiosira spp. and Leptocylindrus danicus were all prominent in the plankton samples, and centric diatoms predominated in the settling particles. Plankton samples contained 0.4 " 0.4 mgrg dw Ž1995. or 1.4 " 1.3 mgrg dw Ž1996. total sterols, with cholesta-5,24-dien-3b-ol Žmean 26% of total sterols., cholest-5-en-3b-ol Ž24%. and cholesta5,22Ž E .-dien-3b-ol Ž13%. chief among these, denoting diatom and zooplankton sources. In settling particles, the prominence of cholesta-5,24-dien-3b-ol Ž24%., cholest-5-en-3b-ol Ž24%., cholesta-5,22Ž E .-dien-3b-ol Ž13%. and 24-methylcholesta5,22Ž E .-dien-3b-ol Ž9%. again suggested mainly marine sources. The sterol composition of plankton and settling particles from different sampling periods showed a high degree of consistency. Higher plant C 29 sterols Žnotably 24-ethylcholest-5en-3b-ol, 9–26%. were prominent in sediments from both inshore and offshore sites. No decreasing trend in total or individual sterols was observed down the 30-cm sediment cores, suggesting good overall preservation. No 5b-stanols such as 5b-cholestan-3b-ol Žcoprostanol. were detected in offshore sediments, plankton or settling particles, with only low levels Ž5b-cholestan-3b-ol max. 4.4%, 5b-cholestan-3a-ol max. 5.1%. in certain inshore sediments. This suggests that raw sewage discharges in rural Newfoundland are being efficiently degraded or dispersed, or that inputs are highly localized. Source apportionation of organic matter in the sediment samples based on sterol composition was attempted. This highlighted the large terrestrial contribution to the sterols in marine sediments Žup to 58% of sterols inshore, 24% offshore. and suggests either degradation or effective recycling of marine sterols. q 2001 Published by Elsevier Science B.V. Keywords: Sterols; Plankton; Sediment traps; Sediments; Lipid biomarkers; Fecal biomarkers

1. Introduction

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Corresponding author. Tel.: q1-709-737-3709; fax: q1-709737-3220. E-mail addresses: [email protected] ŽE.D. Hudson., [email protected] ŽC.C. Parrish., [email protected] ŽR.J. Helleur..

The collapse of the cod fishery in Newfoundland ŽEastern Canada., with its devastating economic and social impacts, provided an impetus for studies to gauge ecosystem health and history in the area. These studies, focusing on the western Trinity Bay

0304-4203r01r$ - see front matter q 2001 Published by Elsevier Science B.V. PII: S 0 3 0 4 - 4 2 0 3 Ž 0 1 . 0 0 0 6 6 - 4

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Fig. 1. Study area and sampling locations in western Trinity Bay, Newfoundland, Canada.

region ŽFig. 1. Žsee also Budge and Parrish, 1998; Favaro, 1998; Parrish, 1998. aimed to determine sources, fates and transformations of organic matter in the marine environment, and the relative contributions of marine vs. terrestrial and natural vs. anthropogenic inputs, by analyzing specific sets of lipid biogeochemical marker compounds Žbiomarkers. ŽSaliot et al., 1991.. Among lipids, sterols are some of the best biomarkers, due to their resistance to degradation ŽSaliot et al., 1991; Quemeneur and Marty, 1992. and their wide variety of structures. A great many individual sterols occur in the oceans, either originating there or from input from terrestrial systems ŽVolkman, 1986.: the identification of 30 or more sterols in settling particles and sediments in various marine environments ŽSmith et al., 1983; Bayona et al., 1989; Harvey and Johnston, 1995. is not unusual. The sterol profile of algae can be characteristic of a particular class, family, genus or even species ŽBarrett et al., 1995.. However, the use of sterol biomarkers is complicated by the variability of the sterol composition which may occur even within certain algal genera ŽPatterson, 1991; Volkman, 1986., the possibility that sterol composition may depend on

algal growth conditions ŽVeron et al., 1996., and transformations that may take place between sources and sinks, such as dealkylation of dietary phytosterols by copepods ŽSerrazanetti et al., 1989.. Nonetheless, certain patterns in the sterol composition of marine samples can provide useful biogeochemical information. A predominance of 4-methyl sterols such as 4a ,23,24-trimethyl-5a-cholest-22Ž E .en-3b-olŽdinosterol. likely indicates a significant dinoflagellate contribution, although certain diatoms have been found to synthesize this sterol ŽVolkman et al., 1993.. While certain algae produce predominantly C 29 sterols, proportionately high abundances of C 29 are, in many cases, still indicative of terrestrial input Že.g., Laureillard and Saliot, 1993; Li et al., 1995.. Parameters such as sterol ratios Že.g., cholest-5-en-3b-olr24-ethylcholest-5-en-3b-ol, cholest-5-en-3b-olr24-ethylcholesta-5,22Ž E .-dien3b-ol and cholest-5-en-3b-olrŽ24-ethylcholesta5,22Ž E .-dien-3b-ol q 24-ethylchol-est-5-en-3b-ol.Li et al., 1995. are best used to establish source or preservation trends within a series of samples Že.g., from different locations along a transect., rather than as absolute indicators of source material. The sterol 5b-cholestan-3b-ol Žcoprostanol. has long been used as a marker of sewage contamination Že.g., Hatcher and McGillivary, 1979; Writer et al., 1995.. While the covariation of 5b-cholestan-3b-ol with algal sterols ŽPocklington et al., 1987. and its occurrence at elevated concentrations in sediments from remote marine locations ŽGrimalt et al., 1991; Colombo et al., 1997. suggest sources besides sewage, the ratio of 5b-cholestan-3b-ol to other sterols such as cholest-5-en-3b-ol or 4a ,23,24-trimethyl-5a-cholest-22Ž E .-en-3b-ol ŽGrimalt et al., 1990; Venkantesan and Kaplan, 1990; Li et al., 1995; Mudge and Bebianno, 1997. or specific fatty acids ŽQuemeneur and Marty, 1992. may still be a reliable means to detect the influence of sewage discharges in marine systems. The 5b-cholestan-3aol to 5b-cholestan-3b-ol ratio can distinguish the feces of certain marine mammals from that of humans ŽVenkantesan and Santiago, 1989.. The present study examined sterols in plankton, settling particulate matter and recent sediments from Trinity Bay, a 100-km long, deep Ž590 m max.. fjord-like bay in Eastern Newfoundland. Using supporting information from floristic analysis of

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the samples, we aimed to assign sterols to specific sources and thus to develop a sterol-based apportionation of the sources of organic material in these compartments of this cold ocean ecosystem. Additionally, 5b-cholestan-3b-ol and related 5b-stanols produced by biohydrogenation were examined to ascertain whether sewage inputs were recognizable in the current ecosystem or in the region’s sedimentary record, as sewage is typically disposed of in Newfoundland bays and harbours with no pre-treatment. This is the first study to examine sterols in all of plankton, settling particles and sediments in a sub-polar ocean ecosystem and where sterol source assignments are directly supported by floristic analysis of the same material.

2. Experimental 2.1. Sampling Sample collection and storage are described in detail in Parrish Ž1998.. Sediment cores Ž30 cm deep. were obtained from an offshore site in Trinity Bay ŽSt-7, Fig. 1. and two inshore sites in the Northwest Arm of Trinity Bay ŽH-1, H-9. with a box corer during the spring and summer of 1994. Moored sediment trap arrays ŽParrish, 1998. were deployed at St-9 and St-7 during 1994 and 1995 at depths of 50, 75 and 100 m, with four collectors on a single array at each depth being deployed and recovered together to allow replicate sampling. Traps were recovered and redeployed several times over the year-round period; those reported here were recovered in July 1994 ŽSt-9. after a 27-day deployment and in April 1995 ŽSt-7. after a 265-day Žoverwinter. deployment. Finally, seven 20-mm mesh plankton net-tows Žvertically from 100 m depth to surface, and horizontally at the depth of maximum chlorophyll fluorescence. were taken at sites St-7, St-9 and H-9 during April and May of 1996. 2.2. Sample preparation Sediment cores were sectioned into 2-cm portions Ždepth. after removal of the outer 1 cm Ždiameter. of

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each core. Sediments contained appreciable amounts of water, and sections were air-dried overnight in low light conditions. Visible debris such as sticks and shells were removed, sediment was ground with a mortar and pestle, and ; 10 g of each section, plus ; 1 g pre-cleaned anhydrous Na 2 SO4 , was Soxhlet extracted for 24 h with 9:1 dichloromethanermethanol in a pre-extracted cellulose thimble. One quarter of the extract, previously evaporated to dryness and stored frozen, was re-dissolved in 1 ml of 2:1 Žvrv. hexanerchloroform by sonication Žice bath, 1 min. for analysis. Sediment trap and plankton tow samples were collected on pre-combusted GFrC glass fibre filters, extracted with 2:1 chloroformrmethanol by a modified Folch procedure ŽFolch et al., 1957; Parrish, 1999., and the lipids recovered in chloroform to give a total lipid extract ŽTLE.. TLEs were stored in the dark under N2 at y20 8C until analysis. The proportion of organic matter in the samples was determined by weighing before and after combustion at 450 8C for 16–20 h Žsee Parrish, 1998.. 2.3. Chemical analysis TLEs Ž100 or 500 ml, depending on availability and anticipated sterol concentration. were evaporated to dryness under N2 , then saponified with 100 ml saturated Ž12% wrv. KOH in methanol Ž75 8C, 1 h.. After the addition of water Ž400 ml., the solution was shaken and extracted with six aliquots Ž100–200 ml each. of 2:1 hexanerchloroform. The pooled extracts were evaporated to dryness under a flow of N2 and derivatized with 50 or 200 ml of bis-N,OŽtrimethylsilyl.trifluoroacetamide ŽBSTFA. containing 1% trimethylchlorosilane ŽTMCS. ŽSupelco, Bellefonte, PA. Ž60 8C, 1 h.. Where necessary, six drops of silylation-grade pyridine ŽSupelco. were added to dissolve all the material before derivatization. Nine sterol standards Ž5b-cholestan-3b-ol, 4,4,14-trimethyl-5a-cholesta-8,24-dien-3b-ol, 24ethylcholesta-5,24Ž28.Ž E .-dien-3b-ol- Steraloids, Wilton, NH, and 24-methylcholest-5-en-3b-ol, 24ethylcholest-5-en-3b-ol, 5b-cholestan-3a-ol, cholest5-en-3b-ol, 5a-cholestan-3b-ol, 24-ethylcholesta522Ž E .-dien-3b-ol- Sigma, St. Louis, MO. plus a coprostanone Ž5b-cholestan-3-one. standard, were

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Table 1 Sterols detected in Trinity Bay samples, in order of increasing retention time Name

Abbreviationa

24-Norcholesta-5,22Ž E .-dien-3b-o1 24-Nor-5a-cholest-22Ž E .-en-3b-o1 5b-Cholestan-3b-ol Žcoprostanol. 5b-Cholestan-3a-ol Ž epicoprostanol. 24-Methyl-27-norcholesta-5,22Ž E .dien-3b-ol Žoccelasterol. Cholesta-5,22Ž E .-dien-3b-ol 5a-Cholest-22Ž E .-en-3b-ol Cholest-5-en-3b-ol Žcholesterol. 5a-Cholestan-3b-ol Žcholestanol. Cholesta-5,24-dien-3b-ol Ždesmosterol. 24-Methylcholesta-5,22Ž E .dien-3b-ol 24-Methyl-5a-cholest-22Ž E .en-3b-ol 24-Methylcholesta-5,24Ž28.dien-3b-ol 24-Methylcholest-24Ž28.-en-3b-ol 24-Methylcholest-5-en-3b-ol 4a-Methyl-5a-cholestan-3b-ol 24-Methyl-5a-cholestan-3b-ol 24-Ethyl-5b-cholestan-3b-ol Žethylcoprostanol. 4a ,24-dimethyl-5a-cholest22Ž E .-en-3b-ol 24-Ethylcholesta-5,22Ž E .-dien-3b-ol 24-Ethyl-5a-cholest-22Ž E .-en-3b-ol 24-Ethylcholest-5-en-3b-ol 24-Ethyl-5a-cholestan-3b-ol 24-Ethylcholesta-5,24Ž28.Ž E .-dien3b-ol Žfucostanol. 24-Ethylcholest-24Ž28.Ž E .-en-3b-ol Žfucostanol. 4a ,23,24-Trimethylcholest-5,22Ž E .dien-3b-ol 24-Ethylcholesta-5,24Ž28.Ž Z .-dien3b-ol Žisofucosterol. 24-Ethylcholest-24Ž28.Ž Z .-en-3b-ol Žisofucostanol. 4a ,23,24-Trimethyl-5a-cholest22Ž E .-en-3b-ol Ždinosterol. Unidentified C 30 ,D5 steratrienol

24nor-26D5,22 E ŽA4. 24nor-26D22 E ŽC4. 5b-27D0 ŽB1. 3a ,5b-27D0 ŽE1. 24Me-27nor-27D5,22 E ŽA7. 27D5,22 E ŽA6. 27D22 E ŽC6. 27D5 ŽA1. 27D0 ŽC1. 27D5,24 ŽA5. 24Me-28D5,22 E ŽA8. 24Me-28D22 E ŽC8. 24Me-28D5,24Ž28. ŽA9. 24Me-28D24Ž28. ŽC9. 24Me-28D5 ŽA2. 4a Me-28D0 ŽD1. 24Me-28D0 ŽC2. 24Et-5b-29D0 ŽB3. 4a ,24diMe-29D22 E ŽD14. 24Et-29D5,22 E ŽA10. 24Et-29D22 E ŽC10. 24Et-29D5 ŽA3. 24Et-29D0 ŽC3. 24Et-29D5,24Ž28. E ŽA12. 24Et-29D24Ž28. E ŽC12. 4a ,23,24triMe-30D5,22 E ŽF13. 24Et-29D5,24Ž28. Z ŽA11. 24Et-29D24Ž28. Z ŽC11. 4a ,23,24triMe-30D22 E ŽD13. 30D5,x,x

Where a commonly used and unambiguous trivial name exists, it is shown in brackets. Letterrnumber designations Žbold text. following the abbreviation refer to Appendix A. Not all sterols were detected in all samples. a Abbreviation of the form x Et-yb-aDb,c indicates a sterol with ethyl substitution Žrelative to cholesterol. at position x, b stereochemistry at carbon y, a total of a carbons, and unsaturation at positions b and c.

used for sterol identification and retention time confirmation. Immediately prior to analysis, the derivatizing agent was evaporated under a flow of N2 . The TMS ethers were redissolved in hexane containing cholestane as an internal standard and analyzed using a Varian 3400 gas chromatograph fitted with a 30 m = 0.25 mm I.D. Ž5% phenyl.polydimethylsiloxane column ŽDB-5, 0.12 mm film; J & W Scientific, Palo Alto, CA.. The temperature program was: 80 8C for 1 min, 50 8Crmin to 235 8C, 5 8Crmin to 305 8C, 305 8C for 15 min, injector 290 8C, FID 315 8C; 20 psi constant He pressure. Each sample was also analyzed by GC-MS on a 25 m = 0.25 mm I.D. CP-Sil 5CB column Ž100% polydimethylsiloxane; 0.12 mm film; DB-1 equivalent; Chrompack, the Netherlands. using a Hewlett-Packard 5890 II GC with a 5971A Mass Selective Detector employing 70 eV electron ionization Žtemperature program: 80 8C for 1 min, 50 8Crmin to 200 8C, 5 8Crmin to 305 8C for 5 min; splitrsplitless injector 290 8C, MS transfer port 220 8C; 1 mlrmin constant He flow.. The mass selective detector was scanned from 35– 530 amu at 1.5 scansrsec. Sterol TMS ethers were identified by their mass spectra and retention times compared with standards and published spectral data ŽGoad, 1991; Jones et al., 1994. ŽTable 1.. Total free sterols Žas distinct from steryl acyl esters and steryl glycosides. in all TLEs were determined by TLC-FID on Chromarods S-III ŽParrish, 1987. using an Iatroscan Mk V ŽIatron Laboratories, Tokyo, Japan.. Each extract was simultaneously analyzed in triplicate on adjacent Chromarods. Cholest5-en-3b-ol was used as an external quantitation standard and hexadecan-3-one as an internal standard. 2.4. Floristic analysis and core dating Subsamples of the plankton tow and sediment trap samples were taken prior to filtration and preserved with Lugol’s iodine and 10% buffered aqueous formaldehyde Ž1 ml each.. A 200-ml aliquot was observed at 200 = magnification under an inverted Zeiss compound microscope. An ocular micrometer and appropriate geometric shapes were used to calculate biovolumes ŽBudge and Parrish, 1998.. Subsamples from the sediment cores ŽSt-7, H-1, H-9. were

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dated using 210 Pb ŽSchell, 1982. ŽMycore, Deep River, Ontario..

3. Results and discussion 3.1. Plankton composition of the samples Abundances of different phytoplankton taxa in the plankton tows and sediment traps are reported in detail by Budge and Parrish Ž1998.. From March to May 1996, centric diatoms are the most abundant algae in vertical Ž100 m–surface. plankton tows. Chaetoceros and Thalassiosira ŽCentrales. each constitute 11% of algal cells in March, with unidentified centric diatoms comprising a further 50% and dinoflagellates Žparticularly Ceratium tripos, 14%. contributing nearly 20%. The centric diatoms Chaetoceros Ž24%. and Leptocylindrus danicus Ž48%. constitute the bulk of the April plankton tow, with Chaetoceros becoming even more important in May Ž91%.. The June plankton tow is unique for large dinoflagellate contribution from C. tripos Ž85%.. Copepod nauplii were also present in many plankton tow samples ŽParrish, 1998.. Diatoms predominated in the sediment traps. In the St-9 traps ŽFig. 1. recovered in July 1994, Leptocylindrus ŽCentrales., Grammatophora ŽPennales. and unidentified centric diatoms each constituted 33% of algal cells. In the St-7 traps recovered in April 1995 after an overwinter deployment, Coscinodiscus ŽCentrales. and Fragillaria ŽPennales. contributed 73% and 22% of algal cells, respectively. The traps also contained minor quantities of zooplankton fecal pellets. 3.2. Plankton The spring diatom increase Žspring bloom. is the major annual primary production event in Newfoundland coastal waters ŽParrish, 1998.. At this time, lipid fluxes Žup to 43 mg my2 dayy1 . are equivalent to those in highly productive upwelling regions ŽWakeham et al., 1984.. Plankton samples contained 0.4 " 0.4 mgrg dry weight total free sterols Žmean " standard deviation, n s 13. during the spring of 1995 ŽApril, May., and

Fig. 2. Ža. Mean sterol composition of Trinity Bay plankton tows collected from Mar–June 1995 and ’96. Mean"SD; ns6 tows. Both vertical and horizontal tows are included. Žb. Sterol composition of individual plankton tow samples. Mean"SD; ns 2.

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they ranged across an order of magnitude Ž1.4 " 1.3 mgrg dw, n s 26. during the spring of 1996 ŽMarch–June., suggesting considerable annual variation. Of the types of samples, plankton tows generally had the simplest sterol composition, with 8 to 14 sterols being identified in each sample ŽFig. 2.. Not all sterols were detected in all samples. C 27 sterols predominated; cholesta-5,24-dien-3b-ol Žmean " SD 22 " 8%., cholest-5-en-3b-ol Ž15 " 4%., and cholesta-5,22Ž E .-dien-3b-ol Ž15 " 8%. were consistently present, as were 24-methylcholesta-5,24 Ž28.-dien-3b-ol Ž11 " 4%., 24-norcholesta-5,22 Ž E .-dien-3b-ol Ž7.9 " 2.6% . and 24-methylcholest-24Ž28.-en-3b-ol Ž6.6 " 2.4%.. A sterol which is likely 24-methyl-27-norcholesta-5,22Ž E .-dien-3bol Žoccelasterol. ŽHarvey and Johnston, 1995. was detected in several samples. While cholesta-5,22 Ž Z .-dien-3b-ol, has an identical mass spectrum and elution behaviour ŽJones et al., 1994. similar to 24-methyl-27-norcholesta-5,22Ž E .-dien-3b-ol, cis22-sterols are rare in marine samples. C 29 sterols found in certain samples consisted mostly of 24-ethylcholesta-5,24Ž28.Ž E .-dien-3b-ol and 24-ethylcholesta-5,24Ž28.Ž Z .-dien-3b-ol and their corresponding 5a-stanols. No C 30 sterols were detected. These compositions are fully consistent with a marine origin with major contributions from diatoms. Cholesta-5,24-dien-3b-ol is the major sterol of several Chaetoceros species ŽGladu, 1989., and cholest5-en-3b-ol is an important component of many others ŽPatterson, 1991.. The C 28 sterols detected, 24-methylcholesta-5,22Ž E .-dien-3b-ol and 24-methylcholesta-5,24Ž28.-dien-3b-ol, are among those often associated with diatoms ŽVolkman, 1986; Patterson, 1991.. Particularly, 24-methylcholesta-5, 24Ž28.-dien-3b-ol is the major sterol of Thallasiosira ŽVolkman and Hallegraeff, 1988., which constituted 11% of algal cells in March 1996, and of many Chaetoceros species ŽPatterson, 1991.. A possible source of 24-ethylcholesta-5,24Ž28.Ž E .-dien-3b-ol is Chaetoceros, in which this sterol has been found Žup to 41%. ŽTsitsa-Tzardis et al., 1993., although 24-ethylcholesta-5,24Ž28.Ž E .-dien-3b-ol and 24-ethylcholesta-5,24Ž28.Ž Z .-dien-3b-ol are normally associated with macrophytic brown algae ŽPatterson, 1991.. As previously noted ŽSection 3.1., Chaetoceros constituted 91% of algal cells in the May 1996 plankton tow, and the sterol composition

of this tow is consistent with its high abundance ŽFig. 2b.. The cholesta-5,22Ž E .-dien-3b-ol present is likely due to zooplankton, as are the C 26 Ž24-nor. sterols ŽSerrazanetti et al., 1989; Volkman et al., 1981.. While C 26 Ž24-nor. steranes have often been identified in diatomaceous sediments ŽHolba et al., 1998., they have not been detected in living diatoms and are known to be present in zooplankton. The absence of C 30 sterols, often prominent in dinoflagellates ŽVolkman et al., 1999. may reflect that the most important dinoflagellate contributions were in June, while all the plankton samples analyzed for individual sterols were taken earlier in April and May. Unfortunately, no TLE of a June plankton tow was available for individual sterol analysis. D5-Stenols are more susceptible to biodegradation in oxic waters than are sterols with saturated steroid nuclei Žstanols. ŽVolkman et al., 1981., and thus the ratio of stanols to their D5-unsaturated analogues Žstanolrstenol ratio. can be a useful indicator of trends in the preservational status of organic matter Že.g., Li et al., 1995.. While de novo synthesis of stanols by certain algae ŽPatterson, 1991; Volkman et al., 1999. means that extensive biodegradation cannot necessarily be inferred from elevated stanolr stenol ratios, low ratios would indicate that neither direct algal input nor bacterial reworking of stenols is contributing appreciable amounts of stanols to the material. Stanolrstenol ratios in our plankton ranged from 0.19 " 0.31 Ž24-nor-5a-cholest-22Ž E .-en-3bolr24-norcholesta-5,22Ž E .-dien-3b-ol. to 0.57 " 0.31 Ž24-methylcholesta-5,24Ž28.-dien-3b-olr24methylcholest-24Ž28.-en-3b-ol., with the corresponding stanols of the sterols cholest-5-en-3b-ol and cholesta-5,22Ž E .-dien-3b-ol being entirely absent. This suggests little bacterial reworking of sterols. While this would not necessarily indicate the preservational status of other compound classes in the material, it does support the inference of good preservational status from low hydrolysisrlipolysis indices ŽParrish, 1998.. 3.3. Settling particles Settling particles collected at St-7 at 50, 75, and 100 m depths in April 1995 after an overwinter trap deployment contained 0.4 " 0.2 mgrg dw total free

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sterols Žmean " SD, n s 7., not significantly different Ž P ) 0.05. from the 0.58 " 0.38 mgrg Ž n s 9. reported for the Laurentian trough ŽColombo et al., 1996., a temperate, deep nearshore region. The concentrations did not differ significantly between the 50 and 100 m arrays Ž P ) 0.05., suggesting that sterols are not lost by conversion to non-sterol molecular species during sinking through the upper water column. Relatively rapid settling rates for particles in this environment Žapproximately 20 mrday. ŽRedden, 1994., in combination with the year-round sub-zero temperatures throughout much of the water column in Trinity Bay, imply little bacterial degradation during sinking. Cholest-5-en-3b-ol Ž17 " 5%, n s 6., cholesta5,24-dien-3b-ol Ž24 " 6%. and cholesta-5,22Ž E .dien-3b-ol Ž10 " 2%. and 24-methylcholesta-5, 22Ž E .-dien-3b-ol Ž9 " 6%. were among the principal sterols in settling particles at St-7 ŽFig. 3.. This is similar to the sterol composition of the 1996 plankton tows and clearly indicates plankton as the main source of the material, even in these overwinter trap deployments. However, unlike in the plankton, 24methylcholest-5-en-3b-ol Ž6 " 2%. and 24-ethylcholest-5-en-3b-ol Ž5 " 3%. were also found. Their absence from the plankton samples suggest possible non-diatom sources in the settling particles, although certain diatoms do produce these sterols ŽVolkman, 1986., including 24-methylcholest-5-en-3b-ol by certain Fragillaria ŽPatterson, 1991.. There were few discernible trends in individual sterol species with depth ŽFig. 3., although the number of species detected increased, particularly between 75 and 100 m. The proportion of 24-ethylcholesta-5,24Ž28.Ž E .-dien-3b-ol decreased with depth. None was detected at 100 m, possibly due to its transformation to 24-ethylcholesta-5,22Ž E .-dien3b-ol during the C-24 dealkylation process by zooplankton ŽSerrazanetti et al., 1989.. This is supported by the presence of 24-ethylcholesta-5,22Ž E .-dien3b-ol and the corresponding stanol 24-ethyl-5acholest-22Ž E .-en-3b-ol at only 100 m depth, although these were not detected at 75 m despite a ) 50% decrease in the proportion of 24-ethylcholesta-5,24Ž28.Ž E .-dien-3b-ol in going from 50 to 75 m. At this depth, the 24-ethylcholesta-5,22Ž E .dien-3b-ol may already have been transformed to cholesta-5,24-dien-3b-ol, a further step in the zoo-

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Fig. 3. Sterol composition of settling particles at St-7 collected April 1995. Mean"SD, ns 2 collectors per depth.

plankton-mediated dealkylation process ŽSerrazanetti et al., 1989.. Cholesta-5,24-dien-3b-ol is particularly prominent at 75 m. Settling particles intercepted at the same depths at St-9, nearer to shore, in July 1994 after a 27-day trap deployment had a similar sterol composition to those from St-7 and to the plankton samples, with cholest5-en-3b-ol Ž19 " 8%, n s 3., cholesta-5,24-dien-3bol Ž9 " 3%., and 24-methylcholesta-5,22Ž E .-dien3b-ol Ž9 " 5%. most abundant. In contrast to the St-7 samples, 24-methylcholest-5-en-3b-ol and 24methyl-5a-cholestan-3b-ol are virtually absent. The similarities between the sterol compositions of the settling particles from both summer ŽSt-9. and overwinter ŽSt-7. sampling periods, and plankton collected during spring, along with sampling over different years, suggest that the sterol composition of any autochthonous, marine input to the sediments is reasonably constant at the present time. Nonetheless, annual variation in phytoplankton assemblages may

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explain discrepancies such as 24-methylcholest-5-en3b-ol and 24-methyl-5a-cholestan-3b-ol being detected in St-7 settling particles but not those from St-9 Žcloser to shore. ŽVolkman et al., 1999.. 3.4. Sediments Surface sediments Ž0–2 cm depth. contained 263–410 mg total free sterolsrg organic matter Ž31– 129 mgrg dw sediment., comprising 2.6–7.5% of total lipids ŽTable 2.. These values are comparable to those reported in other nearshore sediments, including those from the Laurentian Trough Ž1.4–42 mgrg dw. ŽColombo et al., 1997., Buzzards Bay Ž8.4–23 mgrg dw. ŽLee et al., 1977. and the Namibian Shelf Ž164–241 mgrg dw. ŽSmith et al., 1982.. Mean subsurface Ž) 2 cm depth. sterol concentrations did not differ significantly between stations, ranging from 3.7 to 79 mgrg dw in individual horizons. However, subsurface percentage sterol contributions to total lipids decreased in the order St-7, H-9, H-1 Ž7.5%, 3.7%, 1.8%, respectively.. Sediment cores had the most complex sterol compositions ŽFig. 4. of the types of material analyzed,

Fig. 4. Sterol region GCrMS total ion chromatogram for the H9 core Ž2–4 cm depth. TLE. Peaks: 1.24nor-26D5,22 E ; 2.24nor26D22 E ; 3.5b-27D0 ; 4.3a ,5b-27D0 ; 5.27D5,22 E ; 6.27D22 E ; 7.27D5 ; 8.27D0 ; 9.24Me-28D5,22 E ; 10.24Me-28D22 E ; 11.24Me-28D5,24Ž28. ; 12.24Me-28D5 ; 13.24Me-28D0 ; 14.24Et-29D5,22 E ; 15.24Et-29D5 ; 16.24Et-29D0 ; 17.4a ,23,24triMe-30D5,22 E ; 18.4a ,23,24triMe30D22 E ; 19.30D5,x,x . ) s Aliphatic alcohol TMS ethers, in some cases coeluting with sterols. Abbreviations are those given in Table 1.

with 19 or more sterols being identified in all samples. Several peak identifications are tentative, and a few remain identified only as sterols, based on characteristic mass spectral fragments Žesp. mrz 129 for D5 sterols. ŽRahier and Benveniste, 1989.. Their retention times suggest C 28 andror C 29 species. In particular, a species tentatively identified as a C 30 , D5 steratrienol ŽMqs 496, prominent mrz 129. was consistently detected in H-9 and H-1 samples ŽFig. 5, peak 19.. Several alkanol TMS ethers eluted in the sterol region, notably the C 26 , C 28 and C 32 species, with smaller amounts of C 30 . Quantitation of 24methylcholesta-5,22Ž E .-dien-3b-ol was complicated by its co-elution with the C 28 alkanol in many samples. In processing GC-FID chromatograms, the leading edge of the peak representing 24-methylcholesta-5,22Ž E .-dien-3b-ol was split off, along with any shoulder present, prior to integration to attempt to account for any contribution from the C 28 alcohol. Among the major sterols are 24-ethylcholest-5-en3b-ol Ž9%, 15% and 26% at St-7, H-9, H-1, respectively., 24-methylcholest-5-en-3b-ol Ž10%, 8%, 7%., cholest-5-en-3b-ol Ž6.7%, 5.5%, 4.2%., and their stanol analogues ŽFig. 5.. Thus, all three sites contained substantially higher proportions of C 29 sterols than the plankton and sediment trap samples, suggesting a greater terrestrial plant contribution, although these C 29 have been linked to diatom inputs in some marine environments ŽVolkman et al., 1999.. The C 30 4-methylsterols 4a ,23,24-trimethyl-5a-cholest-22Ž E .-en-3b-ol Ž1.8%, 2.2%, 4.6%. and 4a , 23,24-trimethylcholest-5,22Ž E .-dien-3b-ol Ž4.9%, 4.6%, 1.3%., commonly associated with dinoflagellate input, were also detected at all three sites. Distinctive dinoflagellate sterols were not detected in the plankton or settling particles analyzed, possibly due to the dinoflagellate population maximum occurring in summer rather than spring Žin particular, dinoflagellates constituted 85% of the June 1996 plankton sample—Section 3.1.. However, the presence of these sterols in all sediments cores suggests either that dinoflagellate input, when such occurs, is well preserved or that the minor contribution of C 30 4-methylsterols is from diatoms themselves. These sterols have been identified in some NaÕicula ŽVolkman et al., 1993., a diatom genus which is a minor constituent of some phytoplankton communities in this environment ŽBudge and Parrish, 1998..

E.D. Hudson et al.r Marine Chemistry 76 (2001) 253–270

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Fig. 5. Sterol composition of Trinity Bay sediments. Whole core means" SD; n s 8–10 individual horizons.

Cholesta-5,24-dien-3b-ol is completely absent from the sediments, despite being a major component of the plankton and sediment trap samples. Zooplankton and bacterial activity may have converted all the cholesta-5,24-dien-3b-ol present to cholest-5-en-3b-ol ŽSerrazanetti et al., 1992, 1994. prior to burial in the sediments, although no decrease in the proportion of cholesta-5,24-dien-3b-ol in settling particles was observed between 50 and 100 m depth at St-7. Therefore, if this conversion occurred, it would have to have taken place deeper in the water column or at the sediment–water interface. Few other studies have detected the fairly labile cholesta5,24-dien-3b-ol in marine sediments; where detected, it is a minor component Ž0.6% of total sterols, Volkman et al., 1981; 0.46%, Smith et al., 1983..

Certain sterols were not detected at all sites. 24-ethylcholesta-5,24Ž28.Ž E .-dien-3b-ol is absent from H-1 sediments, and may indicate that marine macroalgae are a relatively unimportant contributor to sedimentary sterols at this site, whereas macroalgae are more abundant at H-9 ŽPulchan, 2001.. 24-ethyl-5bcholestan-3b-ol was detected only at H-1, possibly due to livestock manure input ŽNichols et al., 1996. from farming on Random Island. A sterol tentatively identified as 4,24-dimethyl-5a-cholest-22 E-en-3b-ol is likely of algal origin. 3.4.1. Identification of terrestrial sterols— correlation with lignin biomarkers 24-Ethylcholest-5-en-3b-ol is among the major sterols of higher plants ŽAkihisa et al., 1991; Goad,

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1991.. Its prominence in the sediments when none was detected in the plankton samples, and its greater prominence at the inshore sites Ž15 " 5% and 26 " 2% of total sterols at H-9 and H-1, respectively. than at St-7 Ž9 " 3%., is consistent with a terrestrial source for this sterol. However, certain diatoms produce this sterol in appreciable quantities ŽVolkman et al., 1999.. Furthermore, the extent of production may be influenced by growth conditions Že.g., Veron et al., 1996. and thus may be seasonally variable. Finally, several recent studies ŽPearson et al., 2000; Matsumoto et al., 2001. have found 24-ethylcholest5-en-3b-ol to be of marine origin in certain sediments. To determine whether 24-ethylcholest-5-en-3b-ol and 24-methylcholest-5-en-3b-ol Žand their corresponding stanols. could be attributed to terrestrial input in these sediments, individual sterol abundances in nine sediment horizons from H-1 and H-9 were tested for correlation with concentrations of total lignin-derived phenols Žboth absolute and TOC-normalized. and 3,4-dimethoxybenzoic acid methyl ester ŽDMBA., a thermochemolysis-derived lignin biomarker. These data were obtained by Pulchan Ž2001., and all three parameters indicate terrestrially derived material. All correlations were of the Pearson product–moment type and were performed at a s 0.05. Laureillard and Saliot Ž1993. used a similar approach, combining sterol ratios and correlation with terrestrial amyrins, to argue that 24-ethylcholest-5-en-3b-ol in particulate and dissolved matter from an estuarine environment was terrestrial in origin, whereas 24-methylcholest-5-en-3b-ol was planktonic. A significant negative correlation between both cholest-5-en-3b-ol and 5a-cholestan-3b-ol and lignin-derived phenols confirmed that these sterols are marine. Both are rare in higher plants ŽAkihisa et al., 1991.. The ratios of 24-ethylcholest-5-en-3b-ol, of 24-ethyl-5a-cholestan-3b-ol, and of their combined abundances to the sum of the cholest-5-en-3bol and 5a-cholestan-3b-ol abundances show a significant positive correlation with lignin-derived phenols, supporting inferences from their presence in sediments but not plankton, and allowing them to be attributed to a terrestrial source with greater confidence. 24-Methylcholest-5-en-3b-ol was negatively correlated with total lignin-derived phenols, and the

sum of the 24-methylcholest-5-en-3b-ol and 24methyl-5a-cholestan-3b-ol-abundances was negatively correlated with all three lignin-derived quantities, despite the presence of these sterols in sediments but not plankton. Due to this ambiguity, these two sterols cannot be confidently ascribed to either terrestrial or marine sources, as attested to by the lack of agreement among previous studies Že.g., Laureillard and Saliot, 1993; Li et al., 1995.. The presence of 24-ethylcholest-5-en-3b-ol Ž9 " 3% of total sterols. at the offshore site St-7 supports other work which indicates a substantial contribution by terrestrial material to sediments even at these sites Že.g., Budge and Parrish, 1998.. It is more abundant than the traditional indicators of marine algal input Ž24-methylcholesta-5,24Ž28.-dien-3b-ol,24-methylcholesta-5,22Ž E .-dien-3b-ol. and the marine sterols detected in the plankton and settling particles. Its high proportion at H-1 is easily explained by input of terrestrial matter from the major watershed on Random Island, which drains into Hickman’s Harbour, the sampling site. 3.4.2. Sediments— historical (downcore) Õariation At core locations H-1 and H-9, surface sediments Ž0–2 cm. contained significantly more total free sterols Žmgrg OM. than subsurface sediments ŽTable 2., with a 40–60% loss in going from surface to subsurface. This is similar to 66% loss of sterols over the depth of a 30-cm core noted in the Laurentian Trough ŽColombo et al., 1997.. However, at no

Table 2 Total free sterols Žmean"SD. in surface Ž0–2 cm depth; ns 2. and subsurface Ž2–30 cm depth; ns 7. sediments Location and depth

Total free sterols Žmgrg OM.

Free sterols, Ž%. of total lipids

St-7

263"46 a 245"74a 410"101) ,a 167"79 ) ,a 405"17 ) ,a 244"105 ) ,a

7.5"0.3 a 7.5"1.9 a 2.6"0.7 b 1.8"0.8 b 3.7"1.5 b 3.7"1.7 c

H-1 H-9

surface subsurface surface subsurface surface subsurface

Values with different superscripts differ significantly between sites Ž P - 0.05.. ) Means differ significantly Ž P - 0.05. between surface and subsurface.

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site did the percentage contribution of sterols to total lipids differ significantly between surface and subsurface sediments, suggesting that sterols are not degraded faster than other lipid classes. The St-7 core showed evidence of extensive bioturbation, and the lack of a surface–subsurface decrease ŽTable 2. at St-7 is likely due to this homogenizing effect, rather than reflecting better downcore preservation. At H-9, the bottom of the core Ž24–26 cm deep. dates from approximately 1922. 210 Pb dates suggested a far slower sedimentation rate at H-1, with material from 1928 being encountered at just 4–6 cm depth. Assuming equal sedimentation rates lower down, as supported by 14 C dating of another core from the same area ŽE. Burden, pers. comm.., the bottom of the H-1 core dates from ca. 1400 AD. 210 Pb dating was unable to establish a clear age sequence with depth in the bioturbated St-7 core. The high elemental sulphur content of St-7 sediments ŽFavaro, 1998., suggests that they are nonetheless an anoxic environment. Downcore total sterol profiles had minima at 16–18 cm deep ŽH-1., and 8–10 cm deep ŽH-9., regardless of how the sterol

263

content is expressed ŽFig. 6., suggesting declines in productivity or increased fluxes of sterol-poor material at these points. The 210 Pb-estimated sedimentation rates varied by only 15% throughout the H-9 core, implying that increased sedimentation cannot account for these minima. Furthermore, the minimum at 8–10 cm is more pronounced when expressed relative to organic matter content, suggesting a change in the nature of organic material inputs. The presence of these minima at different dates in the two cores suggests some site-specific variations in the biogeochemical history of the area. Few downcore trends in sterol composition could be discerned, suggesting good preservation of the material during burial and no major changes in sources of organic matter in the recent past. At H-9, the combined contribution of typically terrestrial sterols Ž24-ethylcholest-5-en-3b-ol, 24-ethyl-5a-cholestan-3b-ol and 24-ethylcholesta-5,22Ž E .-dien-3bol., has a pronounced minimum at 10–12 cm depth ŽFig. 7a., suggesting a relative decrease in terrestrial input at that time, and a maximum at the bottom of the core Žpre-1920s.. Downcore alkane distributions

Fig. 6. Total free sterol contents of sediments in individual depth horizons at inshore stations. Mean " SD, n s 3 analyses per depth. Vertical error bars indicate the 2-cm depth increments into which the core was sectioned for analysis.

E.D. Hudson et al.r Marine Chemistry 76 (2001) 253–270

264

Fig. 7. Downcore combined percentages of 24Et-29D5 , 24Et-29D0 and 24Et-29D5,22 E at Ža. H-9 and Žb. H-1.

at H-9 ŽFavaro, 1998. reflect the early twentieth century sawmill boom in the area and a re-intensification of woodcutting in the 1950s. Similarly, at H-1, the small maximum in the combined percentages of these sterols at 20–22 cm depth ŽFig. 7b. suggest an increase in terrestrial input, which is corroborated by the abundance in this horizon of the lignin biomarker, DMBA ŽParrish et al., 2000.. The bioperturbed nature of St-7 sediments is consistent with more degradation; in contrast; the fast sedimentation rate at H-9 Žbased on 210 Pb dating, Section 3.4.2. may lead to better preservation. 3.5. Fecal biomarkers No 5b-stanols or stanones were detected in sediment trap and plankton tow samples, nor were stanones detected in the sediments. Small quantities of 5b-cholestan-3a-ol Žup to 4.1%. were present in the bioperturbed St-7 sediments; no 5b-cholestan3b-ol was detected. Colombo et al. Ž1997. reported that biohydrogenation of cholest-5-en-3b-ol to 5bcholestan-3b-ol appeared to be less important in sediments from a site characterized by bioturbation.

Certain core horizons from H-1 and H-9 contained 5b-cholestan-3b-ol andror 5b-cholestan-3a-ol as minor components—a maximum of 4.4% 5bcholestan-3b-ol ŽH-9, 13 cm depth. and 5.7% 5bcholestan-3a-ol ŽH-1, 15 cm.. 5b-cholestan-3b-ol has been found to constitute over 20% of sterols in certain sedimentary horizons far offshore in the Laurentian Trough ŽColombo et al., 1997., where it was attributed to in situ bacterial biohydrogenation. Anoxic sedimentary environments appear to favor the formation of 5b-stanols over 5a-stanols from D5-stenols ŽMeyers and Ishiwatari, 1993.. Lastly, high 5b-cholestan-3a-ol to 5b-cholestan-3b-ol ratios can indicate the feces of baleen whales, rather than of humans ŽVenkantesan and Santiago, 1989.; baleen whales are common in Newfoundland coastal waters in the spring and summer, and this may account for the presence of 5b-cholestan-3a-ol without 5bcholestan-3b-ol at St-7. The ratio of 5b-cholestan-3b-ol to cholest-5-en3b-ol has been used as an indicator of sewage contamination ŽQuemeneur and Marty, 1992; Mudge and Bebianno, 1997.; Quemeneur and Marty Ž1992. have proposed that values of 1 or above suggest appreciable contamination. At H-9, this ratio was

E.D. Hudson et al.r Marine Chemistry 76 (2001) 253–270

0.40 " 0.26 Žwhole core, mean " SD., and did not exceed 0.70 in any single horizon. At H-1, the ratio was 0.21 " 0.25, never exceeding 0.56 and with 5b-cholestan-3b-ol not detected in surface sediments or between 14 and 24 cm depth. The data suggest that, despite the universal practice of discharging sewage into coastal waters from small communities in the Trinity Bay area, the quan-

265

tities discharged are insignificant, or that dispersal and degradation of the material is efficient. Alternatively, a fecal sterol signature in sediments may be highly localized near sewage outfalls, since 5bcholestan-3b-ol is strongly associated with particulate matter ŽPierce and Brown, 1984., and its concentration may therefore drop sharply away from a discharge point. Since the original coring sites were

Fig. 8. Sterols in Trinity Bay plankton, settling particles and sediments according to probable source material Žpercentages of total identified sterols; unidentified sterols were not included.. Sediments, n s 8–10; settling particles, n s 9; plankton, n s 10. Ža. Sources by type of organism Žb. terrestrial vs. marine sources.

266

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not chosen based on distance to sewage outfalls, it is possible that a sedimentary sewage signature is present but restricted to the immediate vicinity of discharge sites. 3.6. Source attribution of sterols in Trinity Bay Based on the preceding discussions of sterols in the plankton, settling particles and sediments, sources of these sterols in each sample type are assigned as noted in Table 3 and apportioned according to their probable origins ŽFig. 8.. Similarly, Colombo et al. Ž1997. detected 14 sterols in sediments in the Laurentian Trough and grouped them into four categories Žphytoplankton, zooplankton, and terrestrial plant sources, with 5b-cholestan-3b-ol reported separately.. Several sterols presented some problems in source assignment. Cholest-5-en-3b-ol Žand 5a-cholestan-3b-ol. is a major sterol in several of the diatom species detected, in many dinoflagellates ŽPatterson, 1991., in zooplankton ŽSerrazanetti et al., 1989.. Furthermore, two of the common macroalgae of the area, Ptiloda serrata and Rhodomela conferÕoides ŽPulchan, 2001. are Rhodophyceae, many of

which contain cholest-5-en-3b-ol as the major sterol ŽPatterson, 1991.. Cholest-5-en-3b-ol and 5acholestan-3b-ol are therefore not included in these source assignments. 24-Methylcholest-5-en-3b-ol and 24-methyl-5a-cholestan-3b-ol are also not included due to the ambiguities discussed in Section 3.4.1. 5b-Stanols, all of which are formed by bacterial biohydrogenation, are grouped separately, in the manner of Colombo et al. Ž1997., although the stenols from which they are derived would originate in one of the other source categories. The contribution of higher plant sterols to the sedimentary material contrasts clearly with the nettow Žplankton. and settling particle samples, to which diatoms and zooplankton are the major contributors of sterols ŽFig. 8a.. Higher plant material makes the greatest contribution at H-1 Ž59% of sedimentary sterols., but even at offshore St-7, at 363 m, higher plant material is still among the greatest contribution Ž30%.. Venkantesan et al. Ž1987. observed typical higher plant sterols in the North Atlantic off the New England coast in sediments from depths greater than 1000 m. Thus, if anthropogenic activities alter the amount of terrestrial input into the marine environ-

Table 3 Attribution of sterols in Trinity Bay samples to various sources of organic matter Source

Sterols attributed to source

Higher plants

24-ethylcholesta-5,22Ž E .-dien-3b-ol,24-ethyl-5a-cholest-22Ž E .-en-3b-ol, 24-ethyl-5a-cholestan-3b-ol, 24-ethylcholest-5-en-3b-ol Phytoplankton 24-methyl-5a-cholest-22Ž E .-en-3b-ol,24-methylcholesta-5,22Ž E .-dien-3b-ol, cholesta-5,24-dien-3b-ol, 4a ,24-dimethyl-5a-cholest-22Ž E .-en-3b-ol,24-methylcholest-24Ž28.-en-3b-ol,24-methylcholesta-5,24Ž28.-dien-3b-ol, 4a ,23,24-trimethylcholest-5,22Ž E .-dien-3b-ol, 4a ,23,24-trimethyl-5a-cholest-22Ž E .-en-3b-ol, 4a-methyl-5a-cholestan-3b-ol Zooplankton 24-nor-5a-cholesta-22Ž E .-en-3b-ol, 24-norcholesta-5,22Ž E .-dien-3b-ol, 24-methyl-27-norcholesta-5,22Ž E .-dien-3b-ol, 5a-cholesta-22Ž E .-en-3b-ol, cholesta-5,22Ž E .-dien-3b-ol Macroalgae 24-ethylcholest-24Ž28.Ž E .-en-3b-ol, 24-ethylcholesta-5,24Ž28.Ž E .-dien-3b-ol, 24-ethylcholest-24Ž28.Ž Z .-en-3b-ol, 24-ethylcholesta-5,24Ž28.Ž Z .-dien-3b-ol Biohydrogenation 5b-cholestan-3b-ol, 5b-cholestan-3a-ol, 24-ethyl-5b-cholestan-3b-ol Marine 5a-cholestan-3b-ol, cholest-5-en-3b-ol, 24-methyl-5a-cholest-22Ž E .-en-3b-ol, 24-methylcholesta-5,22Ž E .-dien-3b-ol, cholesta-5,24-dien-3b-ol, 4a ,24-dimethyl-5a-cholest-22Ž E .-en-3b-ol, 24-methylcholest-24Ž28.-en-3b-ol, 24-methylcholesta-5,24Ž28.-dien-3b-ol, 4a ,23,24-trimethylcholest-5,22Ž E .-dien-3b-ol, 4a ,23,24-trimethyl5a-cholest-22Ž E .-en-3b-ol, 4a-methyl-5a-cholestan-3b-ol, 24-ethylcholest-24Ž28.Ž E .-en-3b-ol, 24-ethylcholesta5,24Ž28.Ž E .-dien-3b-ol, 24-ethylcholesta-24Ž28.Ž Z .-en-3b-ol, 24-ethylcholesta-5,24Ž28.Ž Z .-dien-3b-ol, 24-nor5a-cholest-22Ž E .-en-3b-ol, 24-norcholesta-5,22Ž E .-dien-3b-ol, 24-methyl-27-norcholesta-5,22Ž E .-dien-3b-ol, 5a-cholest-22Ž E .-en-3b-ol, cholesta-5,22Ž E .-dien-3b-ol Terrestrial 24-ethylcholesta-5,22Ž E .-dien-3b-ol,24-ethyl-5a-cholest-22Ž E .-en-3b-ol, 24-ethyl-5a-cholestan-3b-ol, 24-ethylcholest-5-en-3b-ol

The categories AmarineB and AterrestrialB refer to Fig. 8b.

E.D. Hudson et al.r Marine Chemistry 76 (2001) 253–270

ment, this may be reflected in sediments far into the oceans. The assignments of sterols to sources confirm phytoplankton as the major contributor to the plankton tows Ž50% of sterols., although the zooplankton contribution is also substantial Ž34%.. Cholest-5-en-3b-ol and 5a-cholestan-3b-ol not being assigned to specific sources may cause an underestimation of the contribution of marine sources Žphytoplankton, zooplankton, macroalgae. to the sterol pool. Therefore, a further, less specific source assignment, in which sterols were classified only as marine or terrestrial ŽTable 3. was undertaken. This approach leads to smaller, though nonetheless appreciable, proportion Ž24%. of sterols in St-7 being designated as terrestrial ŽFig. 8b.. It is important to note that a quantitative assignment of biomarkers such as sterols to certain sources assumes equal or at least comparable total sterol abundances in all the sources of organic mater Žzooand phytoplankton, higher plants, macroalgae.. Even within taxa, considerable variation can exist. Reported sterol contents span 0.1–0.8% for marine dinoflagellate species ŽMansour et al., 1999., and 91–1354 mgrg dw for various marine phytoplanktonic algae Žincluding Chaetoceros calcitrans, 515 mgrg, and Thalassiosira suecica, 256 mgrg. ŽVeron et al., 1998.. The next stage in the application of this approach would be to correct these using expected or typical concentrations in source organisms, though an additional limitation would be the differing abundance of sterols in different tissues with unequal decomposition rates. Confidence in sterol source assignments could also be increased by the separation of the 24a and 24b epimers of certain sterols ŽMaxwell et al., 1980., although long analysis times preclude doing this routinely.

4. Conclusions The sterol composition of Trinity Bay plankton net-tow material was predominated by the C 27 sterols cholest-s-en-3b-ol, cholesta-5,24-dien-3b-ol, and cholesta-5,22Ž E .-dien-3b-ol, and the C 28 sterols 24-methylcholesta-5,24Ž28.-dien-3b-ol and 24-methylcholesta-5,22Ž E .-dien-3b-ol, all suggestive of marine Žespecially diatom. input. The sterol composition of settling particles and especially sediments

267

was more complex than that of plankton. The terrestrial sterols 24-ethylcholest-5-en-3b-ol and 24-methylcholest-5-en-3b-ol, and their stanol analogues were prevalent in the sediments Žup to 28% for 24-ethylcholest-5-en-3b-ol alone.. Attribution of sterols to certain sources of organic matter in each compartment contrasted plankton and settling particles, in which virtually all input was marine, with sediments, where it was not. In sediments from inshore sites, up to 58% of sterols were terrestrial, and even at offshore St-7, 24% of sterols were terrestrial. This suggests that terrestrial materials, including those introduced by human activity, may become widespread in this marine ecosystem. Examination of 5b-stanols suggested that sewage input is minimal, readily dispersed or highly localized. The simultaneous study of all of plankton, settling particles and sediments from this ecosystem, supplemented by floristic analysis and correlation with lignin-derived phenol biomarkers in the sediments, has led to a more certain attribution of particular sterols in each environmental compartment as being from marine or terrestrial sources; however, this cannot necessarily be generalized to the origin of all organic matter in these materials.

Acknowledgements We thank the crews of the F.R.V. Shamook and the M.V. Nain Banker. We also thank Jeanette Wells and Stewart Parsons for technical assistance, Dr. K. Jerry Pulchan and Yvette Favaro for access to their lignin biomarker and hydrocarbon data, and the National Sciences and Engineering Research Council, Environment Canada’s Tri-Council Eco-Research program, and Memorial University of Newfoundland for financial support. Associate editor: Dr. Stuart Wakeham.

Appendix A Structure of sterols discussed in the text. Lettersr numbers refer to sterols in Table 1. Nuc s sterol nucleus ŽA–F.; R s side chain Ž1–13..

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