Fatty acids in the Ria Formosa Lagoon, Portugal

PII: S0146-6380(98)00049-7

Org. Geochem. Vol. 29, No. 4, pp. 963±977, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0146-6380/98 $ - see front matter

Fatty acids in the Ria Formosa Lagoon, Portugal S. M. MUDGE1*, J. A. EAST1, M. J. BEBIANNO2 and L. A. BARREIRA2 School of Ocean Sciences, University of Wales, Menai Bridge, Bangor, Anglesey LL59 5EY, U.K. and 2 Unidade de Ciencias e Tecnologias dos Recursos Aquaticos, Universidade do Algarve, Campus de Gambelas, 8000 Faro, Portugal

1

(Received 21 August 1997; returned to author for revision 22 October 1997; accepted 20 May 1998) AbstractÐFatty acids in surface sediments were analysed from 59 sites in the Ria Formosa Lagoon, Portugal as part of a wider study on trace organic matter in this system. A total of 170 di€erent fatty acids were quanti®ed as their methyl esters using GC-MS techniques. The TOC in the sediments ranged from 0.15% in sandy areas to 8.3% in muddy regions. The total fatty acids in the sediments ranged between 0.3 to 67.5 mg
gÿ1 the latter accounting for 00.5% of the TOC. Individual compounds, ratios and multivariate statistical methods were used to identify the sources and their spatial variations. The sediments in the region near Armona were rich in phytoplankton biomarkers (e.g. 16:1o7/16.0 and 16 carbon PUFAs), those near Faro and OlhaÄo were comprised of odd chain length fatty acids, their branched derivatives, 18:1o7 and 3-OH compounds; these suggest bacterial biomass linked to the known sewage discharges in these areas. Limited regions of terrestrial in¯uence could be seen through use of the long chain (>22 carbon) saturates. The concentrations of 20:4o6 and 20:5o3 were high in regions of marine production. Multivariate statistical techniques (PLS, PCA and cluster analysis) were used to interpret the data. PCA was found to be the most useful in this case and major axes representing phytoplankton, terrestrial organic matter and bacterial biomass were extracted from the data matrix. The spatial variation of each component could be linked to observations of sewage and phytoplankton input. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐfatty acids, Ria Formosa, lagoon, Portugal, biomarkers, phytoplankton

INTRODUCTION

The Ria Formosa is a large barrier lagoon (55  6 km) in Southern Portugal. A physical description of the lagoon and the temperature and salinity distribution can be found in Newton (1995). There are several inlets from the adjacent Atlantic Ocean leading to a tidal range of between 3.5 and 0.7 m at springs and neaps, respectively. This can account for as much as 50% tidal ¯ushing of the lagoon within one day. The entire region is most productive (Mudge and Bebianno, 1997) with many ®sheries based in the lagoon. The Ria Formosa has increased nutrient loading due to land runo€ and sewage discharges (Newton, 1995; Mudge and Bebianno, 1997) which are exacerbated by reduced tidal exchange in the inner regions although the water quality has deteriorated in recent years due to intense economic activity and tourism (Bebianno, 1995). The freshwater input to the lagoon is small and seasonal with almost no runo€ during the summer months. The water salinity can reach 040 with temperatures of 288C (Newton, 1995). The evaporation *To whom correspondence should be addressed. Tel.: +44-1248-351-151; Fax: +44-1248-716-367; E-mail: [email protected]. 963

of water from the shallow inner regions leads to greater salinities in the lagoon compared to the external ocean waters. More recently, a new inlet has been opened up to the Atlantic Ocean near Faro Beach (sampling site 5). It is hoped that this will improve both the ¯ushing of the system and the access for small boats. Initial observations have indicated that the inlet is rapidly ®lling with sediment and dredging will be needed to keep it open. There have been few studies of the organic contaminants in the lagoonal system (e.g. Mudge and Bebianno, 1997) although some unpublished surveys have investigated the organochlorine compounds present in sediments and suspended matter. This current survey of the surface sediments of the Ria Formosa is the most extensive undertaken to date. This paper presents the data for the fatty acids identi®ed in the lagoon. These lipid biomarkers have been shown to provide good source identi®cation for organic matter in several studies around the world (e.g. Nichols et al., 1982; Grimalt and Albaiges, 1990; Nichols and Espey, 1991). Statistical techniques have been employed to extract and quantify individual sources from complex assemblages.

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S. M. Mudge et al. MATERIALS AND METHODS

Surface sediments samples (top 2 cm) were collected from 59 sites across the Ria Formosa Lagoon (Fig. 1) over a period of 4 days in June 1995. The position of the sites was initially chosen to represent all the major regions of the lagoon and the potential input routes. In the ®eld, the position of each site was identi®ed and recorded using a Garmin GPS-45. Samples were kept in a cool box for return to the laboratory and were extracted within 24 h of collection. Approximately 60 g of each ®eld-moist sediment was re¯uxed in 6% KOH in methanol for 4 h. After cooling, the samples were centrifuged to separate the supernatent from the sediment: the solvent was decanted into a separating funnel and the sterols, fatty alcohols, aliphatic and aromatic hydrocarbons were extracted into hexane. The results of these analyses will be presented elsewhere. The remaining methanolic fraction was titrated to pH 5 with hydrochloric acid and the fatty acids were partitioned twice into 20% DCM in hexane. The solvent fractions were combined, concentrated by a combination of rotary evaporation and nitrogen blow down to dryness. At this stage the lipids were weighed, frozen (ÿ208C) and returned to the laboratories in Menai Bridge for derivatisation with BF3 in methanol to form the methyl esters and analysis by gas chromatography-mass spectrometry (Fisons MD-800). The GC conditions were as follows: separation on a BPX-70 column (SGE), 50 m  0.32 mm ID using a cool on-column injection system and a tem-

perature program of injection at 808C for 2 min; 408C
minÿ1 to 1608C; 0.58C
minÿ1 to 1708C; 108C
minÿ1 to 2508C for 10 min. The MS was used in the EI mode at 70 eV with mass scanning from 45 to 585 m/z over 1 s. The results were collected and analysed using the MassLab software package provided with the instrument. Cod liver oil was used as a retention time standard and 23:0 FAME (Sigma) added just prior to the injection was used to quantify the fatty acids. Compounds were identi®ed through a combination of standards (cod liver oil, mixed PUFAs (Sigma and Supelco) and cis/ trans isomer standards (Supleco) and application to the NIST mass spectral library. The GC-MS analyses provided concentration data for 170 di€erent fatty acids. These included straight-chain saturated, mono and poly-unsaturated FAMEs, branched compounds and several derivatives including 3-hydroxy fatty acids. Several geometric isomers (cis/trans) of the polyunsaturated compounds were also seen. The data set obtained was large and has been reduced by extracting a subset of compounds. The subsequent results are based on the 70 compounds that were present in 20 or more of the sampling sites. The data was analysed spatially through use of the Surfer programme (Golden Software) and much of the data is presented as classed postings on base maps of the lagoon. Concentration data is expressed in mg
gÿ1 dry weight of sediment. Subsamples of the sediments were lightly ground and air dried to constant weight to provide the wet weight±dry weight ratio. They were combusted at

Fig. 1. Location map showing the sampling points in the Ria Formosa. Major towns and sewers are also indicated. Several smaller sewage outlet also occur from small communities along the landward boundary of the lagoon.

Fatty acids in the Ria Formosa lagoon

5008C to determine the loss on ignition as a proxy for the total organic carbon content.

Table 1. The location and bulk properties of the surface sediments samples collected from the Ria Formosa Sample No.

RESULTS AND DISCUSSION

The total organic carbon in the sediments expressed as a percentage of the dry weight ranged from 0.15 to 8.3%. As expected, sediments near the sewage outfalls (see Fig. 1) and in regions of restricted water exchange such as the eastern end of the lagoon had the highest values. Easily discernible gradients could be see in the main Faro Channel leading away from the principal sewer of the region. The total fatty acid concentration ranged from 0.3 mg
gÿ1 in the sandy areas to 67.5 mg
gÿ1 (00.5% of total organic carbon) in the muddy inner regions. In general, sites which were likely to receive sewagederived organic matter had concentrations between 20 and 50 mg
gÿ1. Table 1 indicates the location and bulk parameters of each sample. Composite results The distributions of the monounsaturated and polyunsaturated fatty acids, expressed as a percentage of the total fatty acid concentration, were similar to each other (correlation coecient r = 0.54, signi®cant at P < 0.001, Murdoch and Barnes, 1986) with high values (>30% of total fatty acids) seen near the sheltered western end of the lagoon, at Tavira where the GilaÄo Estuary meets the lagoon and near the large inlet at Armona. The regions where sewage is likely to have a major in¯uence on the sedimentary organic matter, such as the Faro Channel, all exhibited low percentages of monounsaturates and polyunsaturates, typically 5% of the total fatty acids; the saturated fatty acids dominated in these regions. The distribution of branched chain fatty acids, principally iso and anteiso odd chain length compounds, was the inverse of the distribution of the unsaturated fatty acids (correlation coecient r = ÿ 0.35, signi®cant at P = 0.01). High percentages (>20% of total fatty acids) were generally seen at sites close to known sewage discharge points and in regions where ®ne-grain sediments accumulate. Since these compounds are known to be present in higher concentrations in bacteria (e.g. Wakeham and Canuel, 1990), this distribution indicated those areas with signi®cant bacterial biomass. Similarly, odd chain length fatty acids are generally synthesised by bacteria while even numbered carbon compounds are the products of the Krebs Cycle where the chain is elongated in 2 carbon sub-units (Parkes, 1987). The odd/even ratio, the inverse of the carbon preference index of Grimalt and Albaiges (1990), showed a similar spatial distribution as the percentage branched fatty acids reinforcing their link to bacterial biomass.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 60 61

Dry wt% 50.9 64.4 96.0 84.4 84.3 66.0 64.7 81.2 63.0 74.1 69.6 83.5 75.2 72.6 77.0 82.0 68.0 77.3 52.7 78.8 79.0 59.1 85.2 84.3 72.5 64.6 51.9 78.3 70.4 74.9 61.9 55.6 73.8 80.3 58.8 79.9 56.4 59.1 85.0 84.5 80.3 57.9 81.6 56.8 84.5 81.3 81.3 82.3 87.0 76.1 82.0 61.8 87.1 82.2 81.2 70.6 78.0 54.3 74.6

TOC (%) 1.3 0.9 0.4 0.5 0.3 1.3 0.9 2.8 1.4 0.5 1.1 0.5 0.7 0.7 0.8 0.7 0.9 1.0 1.1 0.4 0.9 1.0 0.7 0.7 ND 1.1 1.4 0.6 1.3 0.8 1.6 2.8 ND 2.1 ND 0.8 2.3 1.6 0.2 0.7 0.4 1.1 0.3 0.4 0.3 0.4 ND 0.6 0.5 0.8 0.4 1.3 2.5 3.5 0.1 0.6 0.4 8.3 4.3

Total FA concentration (mg
gÿ1) 67.5 2.1 2.3 0.4 0.4 6.4 6.8 0.6 25.0 0.6 4.5 6.2 15.7 9.5 34.4 8.0 1.4 5.6 45.2 7.9 49.3 12.0 0.6 0.3 15.2 42.0 32.4 2.1 24.6 5.3 21.1 18.9 26.4 19.1 37.6 32.2 26.1 22.1 2.6 11.4 9.8 10.8 32.7 1.7 1.2 0.9 1.3 12.4 2.5 19.0 4.2 34.7 24.8 20.1 14.5 61.4 12.2 33.6 33.6

ND = not determined.

Sulphate reducing bacteria can be characterised by the presence of selected 3-OH fatty acids in their cell walls (Boon et al., 1977) and these compounds can be seen in sediments and suspended matter (Wakeham, 1995). In the Ria Formosa, the highest concentrations of these compounds was seen in regions of high fatty acid content. These were consistent with sewage outfalls. When expressed on a percentage basis, the maxima (>3% of total fatty

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acids) occurred at sites approximately 2±3 km from the sewage outfalls and not adjacent to them. This was the case for four of the ®ve major outlets. Speci®c compounds 14-carbon compounds. The saturated fatty acid, 14:0 was present in all samples. This was not surprising since this fatty acid is among the major lipid components of phytoplankton, especially the Bacillariophyceae (Diatoms) and Prymnesiophyceae (Coccolithophorids) (Reitan et al., 1994) and to a lesser extent in some of the Dinophyceae (Dino¯agellates) (Napolitano et al., 1995). In the Ria Formosa, the distribution appears to be determined by both the proximity to sewage discharges and those areas that have experienced phytoplankton blooms. The former have been shown to be rich in saturated fatty acids. Analysis of biota has indicated that 14:1 fatty acids can be found in Cyanobacteria (Caudales and Wells, 1992; Caudales et al., 1993), marine mammals (Kakela and Hyvarinen, 1993) and certain yeasts (Sajbidor et al., 1994). Within the Ria Formosa, the distribution of this fatty acids appear consistent with the likely development of blue± green algae mats especially at the western end of the lagoon. The water circulation is somewhat restricted in this location and the surface sediments appeared stabilised by algal growth. At most other sites, the 14:1 fatty acid was present in trace amounts only. 16-carbon fatty acids. In over 90% of the sites sampled, the unbranched, saturated 16-carbon molecule (16:0) was the principal fatty acid measured. This was not surprising given that it is the major fatty acid in many organisms such as phytoplankton (Reitan et al., 1994), higher plants (e.g. Rao et al., 1990) and is present in waste waters discharged from sewage plants (Quemeneur and Marty, 1994). The distribution of 16:0 within the lagoon mirrored that of the TOC which in turn was linked to the sewage loading (Mudge and Bebianno, 1997) although elevated concentrations also coincided with regions of ®ne-grain sediment accumulation. In this study, three monounsaturated 16-carbon fatty acids (16:1o5, o7, o9, all cis) were identi®ed. The 16:1 fatty acid is relatively common in marine algal species (e.g. Reitan et al., 1994; Berge et al., 1995) although the 16:1o5 isomer is less common and has been identi®ed in some brown macroalgae (Khotimchenko, 1995). In this study, this fatty acid reached 2.3% of the total fatty acid concentration in contrast to the o9 and o7 isomers which achieved 7.3 and 29.9%, respectively. Generally, the o5 isomer was a greater proportion of the total fatty acids in the sediments to the east of the lagoon although the maximum was located in a sample from the west end of the lagoon (site 7).

The major monounsaturated 16-carbon fatty acid was 16:1o7 re¯ecting its abundance in phytoplankton, especially diatoms (Berge et al., 1995; Skerratt et al., 1995; Suzuki and Matsuyama, 1995). This fatty acid contributed greater than 5% of the total fatty acids in the majority of samples taken. The other 16:1 isomer present in appreciable quantities was 16:1o9; this compound has been found to predominate in several species of heterotrophic marine bacteria (Bertone et al., 1996). This fatty acid made up greater than 1% of the total fatty acids only in the west end of the lagoon (with one exception). This pattern of distribution was di€erent to the more general markers for marine bacteria (percentage branched and the odd/even ratio). Several polyunsaturated 16-carbon fatty acids were identi®ed in the lagoonal sediments. When summed, these reached 2.3% of the total fatty acids in the region adjacent to Armona. This was consistent with the maximum value of the diatom marker using the ratio between 16:1o7 and 16:0 fatty acids. This ratio has been used as an indicator for diatoms in suspended particulates (Skerratt et al., 1995; Parrish et al., 1995). In the Ria Formosa, the ratio 16:1o7/16:0 (Fig. 2) indicates regions of enhancement (>0.75) around the large entrance at Armona and in the sheltered western end of the lagoon. Two isomers of 16:2 were identi®ed (bond positions not con®rmed) together with a 16:3 and 16:4 fatty acid. These compounds together with other 18-carbon fatty acids are indicative of algae, especially green algae (Wakeham and Canuel, 1990; Khotimchenko, 1993; Floreto et al., 1996). In most cases in the literature, however, the position of the double bonds was not determined. It is possible to assign the position of double bonds with some polyenoic fatty acids through use of selected fragments in the mass spectra (m/z 108 for o3, m/z 150 for o6 and m/z 192 for o9; Poulos et al., 1986; Vysotskii and Svetashev, 1991). While this works for 18-carbon fatty acids, this procedure does not appear to work for 16-carbon compounds, although this was not conclusively shown. 18-carbon compounds. As with both the 14- and 16-carbon equivalents, the 18:0 saturated fatty acid was present in the highest concentration in sediments near the sewage outfalls, their regions of in¯uence, and areas of ®ne grain sediment accumulation. Of the three monounsaturated 18:1 fatty acids identi®ed, the 18:1o9 (oleic acid) distribution was coincident with the 18:0 although the concentration decreased more rapidly away from the sewage discharge points. A good example of this can be seen in the main shipping channels leading towards Faro and OlhaÄo. In the Faro channel, high concentrations of 18:0 were measured near the sewage works and these decreased exponentially with distance towards the sea. A linear decrease was also seen in the OlhaÄo

Fig. 2. A classed posting of the 16:1o7/16:0 ratio, a marker for diatoms, in the surface sediments. High values can be seen near Armona where the majority of the seawater exchange takes place.

Fatty acids in the Ria Formosa lagoon 967

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S. M. Mudge et al.

channel although the absolute concentrations were less than for Faro. Near the con¯uence of these two channels (sites 25 to 27, 6.5 to 9 km from Faro sewer), the concentrations increase to values observed at the sewer. The total fatty acid concentrations were also high in this vicinity and it is likely that organic (sewage) rich materials were collecting at this point. The dynamics of the water movement through the lagoon are complex but much of the water that enters the major inlet at Armona exits through the Faro outlet (Newton, 1995). Another isomer of 18:1 with the double bond in the o7 (D11) position is classically used as a biomarker for bacterial activity (e.g. Bradshaw et al., 1991). This fatty acid can account for 10.7% of the total fatty acids in a microbial mat and 030% of purple bacteria in a shallow water hydrothermal system (Kharlamenko et al., 1995). Other workers (e.g. Currie and Johns, 1988) have found similar proportions in bacteria. In the Ria Formosa, the concentrations of this fatty acid were strongly correlated to other bacterial markers, notably the total concentration of odd-chain and total branchedchain fatty acids (r = 0.76 and 0.73, respectively, df = 57). There was a relatively poor correlation to the total organic carbon content suggesting that this alone was insucient to lead to an increase the concentration of this bacterial biomarker. Interestingly, there was a poor correlation with the 3-OH fatty acids, usually considered as markers for the sulphate reducing bacteria. Another 18:1 isomer was identi®ed in several samples (18:1o11) albeit at low concentrations (maximum = 0.11 mg
gÿ1). The spatial distribution was somewhat erratic with little obvious correlation to speci®c regions of the lagoon. Several polyunsaturated 18-carbon fatty acids were identi®ed in the Ria Formosa including 18:2o6, 18:3o3 and 18:4o3. Of these, the 18:3, 18:4 and 18:5 fatty acids are generally considered to be of algal origin whereas the 18:2 (linoleic acid) is not usually present in high concentrations in either micro or macro algae. However, some members of the Chlorophyceae do contain appreciable amounts (Dunstan et al., 1992). Linoleic acid does constitute a major fraction of several vegetable oils used in cooking and food processing (Vergroesen and Crawford, 1989) and also domestic waste waters (Quemeneur and Marty, 1994). As a consequence, 18:2o6 can be found in marine sediments close to regions of sewage disposal (e.g. Nichols and Espey, 1991) and has even been suggested as a tracer for such material in near shore systems. In the Ria Formosa surface sediments, high concentrations of 18:2o6 (all cis) can be seen at or near sites of sewage discharge especially sites 33 to 36 near the Faro outfall. When expressed on a percentage of the total fatty acids, regions of ®ne grained

sediment accumulation also stand out. The concentration correlates well with other bacterial biomarkers such as branched chain and odd carbon numbered fatty acids (r = 0.99 for 13:0). This was also con®rmed through multivariate statistical techniques (see below). In some samples, trace amounts of cis/trans and trans/cis 18:2o6 isomers could be identi®ed although they did not contribute signi®cantly to the total fatty acid concentrations (<0.1%). The more unsaturated 18-carbon fatty acids were present in signi®cant quantities in several sites and overall each averaged 01% of the total concentration. Linolenic acid (18:3o3) was present as a small proportion of the total fatty acids at most sites although in the region near Armona, this fatty acid contributed >1.5% of the total. This co-varied with the 16-carbon PUFAs. Together with 18:4o3, this fatty acid is present in several genera of marine phytoplankton (Hama and Handa, 1992; Reitan et al., 1994) except, seemingly, diatoms (Berge et al., 1995; Suzuki and Matsuyama, 1995) which are characterised by the 16:1o7 fatty acid. Not-withstanding the potential for di€erential degradation of these unsaturated fatty acids, it may be possible to use the ratio between the di€erent compounds as an indicator of phytoplankton type. A plot of the ratio between the 16:1o7 and (18:3o3 + 18:4o3) indicates that only in a single site did the 18 carbon PUFAs dominate; at all other sites the ratio was in excess of 1. In a few sites near Tavira, OlhaÄo and the western end of the lagoon, the ratio exceeded 10 suggesting a strong diatom signal. Around Armona, where all of the phytoplankton markers were present, the ratio was between 2 and 5 indicating a mixed phytoplankton population but biased towards diatoms. This is consistent with the observed phytoplankton species observed in the lagoon. The 18:5o3 fatty acid was detected in trace amounts apparently randomly across the lagoon. This fatty acid has been identi®ed as a major component of Coccolithophores (Pond and Harris, 1996) and rarely in green microalgae (Dunstan et al., 1992). No conclusions could be drawn from the limited occurrence in the Ria Formosa. 20-carbon compounds. Several 20-carbon fatty acids were identi®ed in the lagoon including 20:0, 3 isomers of 20:1, 20:2, 20:3, 20:4 and 20:5. The 20:1 fatty acids are widely found in marine animals such as zooplankton and ®sh (Ota et al., 1995; Albers et al., 1996). The mean concentration in the Ria Formosa Lagoon was low (0.11 and 0.10 mg
gÿ1 for the o9 and o7 isomers, respectively) and the highest values were located near regions of phytoplankton productivity as determined from the other fatty acid biomarkers. These regions around the OlhaÄo channel and Armona are extensively used for the culture of shell®sh.

Fatty acids in the Ria Formosa lagoon

The 20:2 and 20:3 fatty acids have been reported in some brown macroalgae such as Sargassum sp. (Khotimchenko, 1991) although the occurrence represented a small percentage of the total fatty acids. In the lagoon sediments, the 20:2 and 20:3 fatty acids were present in low concentrations (means 0.019 and 0.031 mg
gÿ1, respectively). One of the major fatty acids identi®ed in the Ria Formosa lagoon was 20:4o6, which had the 5th highest mean concentration of all fatty acids identi®ed (mean 1.53 mg
gÿ1). The distribution of this compound was similar to that of 20:5o3 which was the 4th most concentrated fatty acid with a mean concentration in the sediments of 2.29 mg
gÿ1. In a cluster analysis (see below), these two compounds were linked at 095%. These fatty acids can be found in most marine organisms either due to synthesis or consumption of food; several species of red and brown unicellular and macroalgae (Dembitsky et al., 1991; Banaimoon, 1992) and diatoms (Fahl and Kattner, 1993) contain substantial quantities of these fatty acids. Higher organisms feeding on these algae are therefore likely to contain such fatty acids (Jonasdottir et al., 1995) or may synthesise them from 18-carbon pre-cursors (Desvilettes et al., 1997). 22-carbon compounds. Although small quantities of 22:1o11 were found in 26 of the sites, the mean concentration was only 0.02 mg
gÿ1 and considered a relatively minor constituent of the sedimentary fatty acids. Signi®cantly greater amounts of 22:5o3 (mean = 0.16 mg
gÿ1) and 22:6o3 (mean = 0.39 mg
gÿ1) were measured. These two fatty acids were strongly correlated (r = 0.999) to the 20:5o3 and therefore, their spatial distribution was similar. These fatty acids have a similar biological origin (e.g. Graeve et al., 1994) and appear to derive principally from phytoplankton. In some copepods, the fatty acid pro®le is directly related to their diet (Kattner et al., 1994) and has been used to determine the food type in seasonal studies (Parrish et al., 1995). Long chain saturated compounds. Fatty acids with chain lengths up to C28 were quanti®ed in the sediment samples. The longer chain moieties (C24+) are characteristic of terrestrial and higher plant organic matter (Currie and Johns, 1988). The latter category includes sea grasses (Nichols et al., 1982) and it may be possible to di€erentiate between these two potential sources through use of the fatty alcohols. This will be investigated elsewhere. The longer chain saturates co-vary signi®cantly compared to the shorter chain (
969

ment plants. A good example of this can be seen in the Faro channel. Odd chain length compounds. In general, odd chain length fatty acids are derived from bacterial sources (Rajendran et al., 1997). The iso and anteiso C15 fatty acids have been used together with the C17 and other compounds (Wakeham and Beier, 1991) as biomarkers for anaerobic bacteria in particulate organic matter from the Black Sea. Several branched fatty acids were identi®ed in the Ria Formosa from C11 to C17 saturates. In addition, other bacterial biomarkers such as two isomers of 17:1 were present. The highest concentrations of these compounds was found in the regions close to sewage discharge points in Faro and OlhaÄo; signi®cantly lower concentrations were found in regions that have shown elevated phytoplankton or terrestrial organic matter. Multivariate statistical analysis Recent literature regarding lipid biomarkers has highlighted the usefulness of multivariate statistical techniques in extracting the maximum information from complex mixtures of compounds (e.g. Yunker et al., 1995; Mudge and Norris, 1997). While individual compounds and ratios using two or more fatty acids can yield information regarding the origin of the organic matter in the sediments, multivariate statistical methods are able to use more of the data in one analysis. This more holistic approach can be very useful but care needs to be exercised in explaining the results and knowledge of the likely origin of the compounds is needed. With this data, several multivariate techniques were investigated and the results of cluster analysis of both observations and variables together with principal components analysis (PCA) are presented below. The Minitab statistical program was used in both cases. Prior to analysis, the raw concentration data were transformed to proportion data to remove the e€ects of grain size and concentration that would appear as the major controlling factor in the subsequent analysis. Other transformation methods were also investigated although simple proportions were found to be adequate. PCA requires less variables (compounds) than observations (sites) and a reduced set of 58 compounds was compiled by discarding those with the largest coecients of variation (Table 2). These were essentially the ones present in the least number of samples. Cluster analysis of variables The results from cluster analysis (single linkage) can be best viewed as a dendrogram (Fig. 3). The degree of similarity (y-axis) between compounds can be seen by their linkage. The technique groups together compounds that co-vary within the 59 samples. In this case, distinct groups of marine,

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Table 2. The fatty acids chosen for inclusion in the multivariate analyses. The remaining samples were rejected on the basis of their coecient of variation 10:0 br 11:0 11:0 br 12:0 12:0 iso 13:0 anteiso 13:0 13:0 br 14:0 14:0

14:1 iso 15:0 anteiso 15:0 15:0 iso 16:0 anteiso 16:0 16:0 br 17:0 16:1o7 16:1o9

iso 17:0 16:1o5 anteiso 17:0 16:2 3-me 17:0 17:0 16:2 17:1 17:1 16:3

bacterial, marine and bacterial, terrestrial and phytoplankton derived fatty acids can be seen (Fig. 3). The terrestrial cluster is comprised entirely by long chain (>23 carbon) saturated fatty acids. In contrast, the phytoplankton cluster contains 16:1o7 together with a series of poly-unsaturated compounds. Two distinct clusters of marine and bacterial fatty acids are apparent in the data along with a cluster containing compounds of mixed origin. These three clusters link together at a similarity greater than 90%. Several fatty acids do not appear to form readily describable clusters and these link individually to the other compounds rather than as sets. Cluster analysis of observations When the observations are clustered, relatively few distinct groups can be seen in the data. One cluster was comprised mainly of sampling sites

16:3 16:4 18:0 18:1o11 18:1o9 18:1o7 18:2o6 19:0 18:3o3 20:0

18:4o3 20:1 20:1o9 20:1o7 20:2 21:0 20:3 20:4o6 22:0 22:1o11

20:5o3 24:0 24:1 22:5o3 22:6o3 25:0 26:0 28:0

from the OlhaÄo region whereas the Faro sites, while appearing close together along the x-axis, are not as signi®cantly linked to either themselves or the OlhaÄo group. Three sites which have been identi®ed as having relatively high concentrations of phytoplankton fatty acids (9, 43 and 44) are among the least well correlated sites. These results are not unexpected as there were no clear end-members from which organic matter could be mixed along a gradient as may be found in estuarine systems. Principal component analysis The calculated principal components (PCs) can be visualised by plotting against each other in turn and viewed as two dimensional scatter plots; the loadings of each fatty acid on individual PCs can be plotted in a similar manner which facilitates easier understanding of the sources ascribed to each PC.

Fig. 3. A dendrogram, showing the degree of similarity between the di€erent fatty acids (variables). Blocks of compounds, nominally from the same source, have been highlighted.

Fatty acids in the Ria Formosa lagoon

The loadings on PC1 (33.5%) and PC2 (12.9%) can be seen in Fig. 4(a). Compounds which are phytoplankton markers such as polyunsaturates and 16:1o7 loaded negatively on PC1 while the bacterial or sewage markers such as branched or odd chain length compounds had positive loadings. A similar axis with marine derived short chain fatty acids loaded positively and long chain moieties loaded negatively can be seen within PC2. The ®rst two PC projections are shown in Fig. 4(b). From the loading data, those samples which project towards the top left (samples 43, 44 and 47) contain the greatest amount of phytoplankton derived organic matter while the somewhat larger group

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towards the centre right are most in¯uenced by sewage. Relatively few sites project towards the bottom of the ®gure indicating terrestrial inputs. This is not unexpected since riverine input is somewhat limited. In general, these results con®rm the assignments made through individual compounds and ratios detailed above. The two ®rst principal components are plotted spatially in Fig. 5. The data are con®ned to two classes which show those that project negatively and those that project positively. Figure 5(a) with PC1 shows the regions that were rich in phytoplankton (grey spots) clearly distinguished from those that are contaminated by sewage (black

Fig. 4. (a) A two-dimensional scatter plot of the loadings on the ®rst two principal components labelled by the compound name. (b) A plot of the ®rst two principal components for each sampling site. Phytoplankton markers can be seen in the top left of the diagram, bacterial (sewage) markers cluster towards the right and an axis of short chain to long chain saturates (marine to terrestrial) can be seen diagonally.

S. M. Mudge et al.

Fig. 5(a).

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Fig. 5. A spatial classed posting of (a) principal component 1 and (b) principal component 2 indicating those sites which project positively or negatively.

Fatty acids in the Ria Formosa lagoon 973

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spots). PC2 [Fig. 5(b)] indicates the essentially marine nature of the samples with several of the sewage e€ected sites also having terrestrial biomarkers. This is probably due to the discharge of runo€ through the same sewage system. Partial least squares (PLS) path modelling The grouping of the sewage e€ected sites allowed these data to be used as X-block (signatures) inputs to a PLS modelling analysis (Geladi and Kowalski, 1986; Naftz, 1996). Sewage e€ected sites were close to known outfalls and these can be seen in the PCA [Fig. 5(b)]. Nine sites were chosen as the signature block and the remaining sites were used as Y-block sites and the extent of sewage contamination within these latter sites can be assessed. This was achieved using the SIMCA-P program from Umetri (Sweden). The choice of signature samples was investigated but there were insucient examples rich in phytoplankton to provide meaningful results. This technique works best if there are clearly discernible end members (Yunker, personal communication). This would be the case in an estuary, but is not the case in this lagoon where the freshwater runo€ is insigni®cant compared to the seawater exchange. Samples rich in certain classes of organic matter (e.g. phytoplankton) were patchy in the lagoon rather than distributed along a gradient. The plot of the X-block (tl) and Y-block (ul) projections was essentially linear with no compounds signi®cantly distant from the line of agreement. The ®rst PC of the X-block data explained 95% of the variation with 2% explained by the second PC. For the Y-block data, PC1 explained 79% and PC2 explained 4% of the variation. The sample projections for the Y-block data (p1 and p2) indicate that most of the sites had signatures similar to that of the sewage signature with a gradient towards those sites principally in¯uenced by phytoplankton. In reality, it would appear as if the sewage was con®ned to the regions adjacent to the discharge points although ®ne-grained ``organic rich'' sediments could be seen at other sites as well. In this particular type of system where gradients from 100 to 0% ``signature'' were not seen, it is dif®cult to ascribe percentage contributions to the sediment using fatty acids alone and a more complex assemblage of compounds may be needed.

SUMMARY OF SOURCES OF ORGANIC MATTER TO THE RIA FORMOSA

The distribution of fatty acids in the Ria Formosa suggest there are several sources of organic matter in the lagoon. These include domestic and industrial wastes (sewage), marine planktonic and benthic production, terrestrial materials and bacteria. While the individual and grouped fatty

acid data describes the spatial distribution of these sources, the use of PCA and PLS multivariate statistical methods greatly simpli®es the description process, providing the loadings can be adequately understood. The magnitude of the various organic contributions varies greatly across the lagoon. The terrestrial organic matter is con®ned to sites near the seasonal rivers according to the data. In general, long chain length fatty acids (>C20) are produced by higher plants whereas short chain fatty acids are derived from both marine and terrestrial sources (Currie and Johns, 1988). The spatial distribution of the ratio of long to short fatty acids indicated those areas that receive freshwater runo€ tend to have higher values of this ratio. Four of the ®ve main rivers can be identi®ed in this manner with ratios in excess of 0.75. These results suggest that these areas receive terrestrially derived organic matter and that there is little redistribution of this material through the lagoon. This can also be seen in the principal component data [Fig. 5(b)]. One of the major sources of organic matter appears to be from phytoplankton. The ratio 16:1o7/16:0 (Fig. 2) indicates regions of enhancement (>0.75) around the large entrance at Armona and in the sheltered western end of the lagoon. It can be surmised from this data that diatom blooms have occurred in these regions previously and a record of this can be seen in the surface sediments through use of this ratio. The change in the dominance between diatom to mixed communities of phytoplankton is indicated by the increasing importance of 18-carbon PUFAs in the inner regions of the lagoon. This was con®rmed qualitatively by comparison of plankton trawl samples within the lagoon by other workers. There is little literature on the location and extent of phytoplankton blooms within the lagoon but Sprung (1994) has suggested that primary productivity is generally low (40 g C mÿ2 aÿ1) even though secondary production of ®sh and shell®sh was high. However, Sprung (1994) observed extensive green macroalgal mats over the autumn to spring period. Domestic sewage is comprised of several fatty acids derived from many potential sources. Oleic acid is the principal fatty acid in several higher plants especially olives and olive oil, the major vegetable oil used for cooking in these southern European countries. Its presence in sewage may be from its use in domestic cooking and the restaurant trade. The rate of decrease in concentration in the surface sediments away from sewage sources was much greater for 18:1o9 compared to 18:0. This may be because there are a whole range of sources for 18:1o9 leading to little enhancement generally or its more labile nature leading to a more rapid breakdown. The overall concentration of 18:1o9

Fatty acids in the Ria Formosa lagoon

was approximately a quarter of the 18:0 concentration in these regions. Spatially, the 18:1o7 is present in elevated concentrations near the sewage discharge sites but not directly at them. This may be due to di€erent fatty acid signatures between the bacterial populations around these sites. Similarly, when expressed as a percentage, this fatty acid appeared to be only a minor component of sediments near these outfalls. This was due to the high concentrations of unsaturated fatty acids present in the discharges themselves. Only at sites greater than 3 km from the discharge points did this fatty acid contribute more than 5% of the total fatty acids. In the Ria Formosa lagoon, the concentrations of fatty acids re¯ect the sediment grain size with low values in the sandy areas and much greater values in the muddy regions. Phytoplankton biomarkers were found in elevated proportions over several sites with sandy substrates compared with greater sewage and marine derieved fractions in the muddy regions. The benthic production is generally high in this lagoon and can be inferred from the substantial shell®shery operating in the muddy regions. Examination of the sediments in these muddy areas shows them to highly populated with several species of marine worms. CONCLUSIONS

These results indicate that through use of selected compounds and/or ratios and especially multivariate statistical approaches, regions with distinct organic matter signatures can be identi®ed. The Ria Formosa lagoon has been under increasing pressure recently through increases in tourism and economic development. The restricted water exchange through the narrow channels has led to the accumulation of contaminants in some regions. The organic matter can be seen in the sediments through use of these biomarkers and on this basis, the lagoon can be divided in to several regions using the above classi®cation techniques. (1) The OlhaÄo channel and Armona area with signi®cant phytoplankton productivity. These results are consistent with observations of blooms in the Ria Formosa (Newton, 1995). The data are also consistent with the observed species distributions (Barbosa, personal communication) where large pennate diatoms have been noted around this main inlet from the sea and a mixed community of smaller species are found within the lagoon. This di€erence was clearly demonstrated by this study. (2) Limited terrestrial runo€, probably only during the winter, to selected sites near the landward boundary of the lagoon. Some input appears to be related to the sewage discharges as well since much of the terrestrial runo€ is directed through the sewage system. This type of organic matter does

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not appear to mix readily with the rest of the lagoonal sediments and so remains con®ned to the inner sites. The dynamics of the lagoon are such that strong mixing does not occur in these sites. (3) Bacterial production in regions with sewage discharges. These areas were con®ned to the main channels around Faro and OlhaÄo. This has already been reported for the OlhaÄo channel (Mudge and Bebianno, 1997). The PCA and PLS results suggest that many of the sites within the lagoon contain sewage derivied materials but the concentrations were small except for the channel system. (4) The other regions behind the barrier islands have mixed sources of organic matter. In these regions the ¯ushing is less restricted than in the channelled wider sections. Small sewage outlets also lead to localised ``hot spots''. In these sheltered regions, seagrasses do occur which may also add to the already diverse sources. At present the lagoon is beginning to experience poor water quality and continued discharges of organic matter and sewage will only increase these problems and also fuel the blooming of toxic phytoplankton. These results indicate the current distributions and should be used to plan the best disposal options for anthropogenic organic wastes. Associate EditorÐG. A. Wol€ AcknowledgementsÐThe travel associated with this work was supported by a grant from the British Council under the Treaty of Windsor Scheme. The analyses were conducted on a GC-MS purchased with assistance from HEFCW. The authors would like to thank Alice Newton for helpful discussions during the interpretations of this work and Mark Yunker for advice on PLS methods. REFERENCES

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