Fatty acids as trophic markers of phytoplankton blooms in the Bahía Blanca estuary (Buenos Aires, Argentina) and in Trinity Bay (Newfoundland, Canada)

Pergamon

PII: SO305-1978(97)OOOS3-7

BiochemicalSystematics and Ecology, Vol. 25, No. 8, pp. 739-755,1997 © 1997 Elsevier Science Ltd

All rights reserved. Printed in Great Britain 0305-1978/97 $17.00+0.00

FattyAcids asTrophic Markers of Phytoplankton Blooms in the Bahia Blanca Estuary (Buenos Aires, Argentina) and in Trinity Bay (Newfoundland, Canada)

GUILLERMO E. NAPOLITANO,*¶ RICARDO J. POLLERO, t ANA M. GAYOSO, t BRUCE A. MACDONALD§ and RAYMOND J. THOMPSONII

*Environmental Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6038, U.S.A.;

TINIBIOLP, Facultad de Ciencias ML=dicas, Universidad Nacional de La Plata, La Plata 1900, Argentina; ~tCentro Nacional Patagbnico (CONICET), Puerto Madryn, Chub~t, Argentina;

§Biology Department, University of New Brunswick, Saint John, New Brunswick, E2L 4L5, Canada; IIOcean Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland, A1C 5S7, Canada

Key Word Index--Placopecten magellanicus; phytoplankton bloom; zooplankton; fatty acids; lipids; food webs. Abstract--The fatty acid compositions of phytoplankton and major primary consumers were analyzed during the development of seasonal algal blooms in the Bahia Blanca estuary, situated on the southern coast of the province of Buenos Aires (Argentina), and Trinity Bay, at Sunnyside, on the eastern coast of Newfoundland (Canada). Primary consumers in the Bahia Blanca estuary were zooplankton dominated by the calanoid copepod Acartia tonsa. At Sunnyside, the primary consumers were the sea scallop Placopecten magellanicus, an ecological and economical important benthic bivalve. The study shows that in spite of obvious differences between the two environments and the analytical approaches employed in each case, the analyses of fatty acid biomarkers can provide relevant ecological information. The fatty acid composition of the lipids of Bahia Blanca phytoplankton (high concentrations of the fatty acids 14:0, 16:10}7, 16:40}1, and 20:50}3) reflected the presence of diatoms as a major component throughout the bloom. Fatty acid markers of the post-bloom phytoplankton in Bahia Blanca indicated a decline of phytoplankton biomass, and a relatively high input of detritus and terrestrial plant materials to the particulate organic matter of the estuary. Linoleic acid (18:20)6), a typical "terrestrial" fatty acid, was conspicuous in the lipids of the post-bloom particulate matter of the Bahia Blanca estuary; 18:20)2 was subsequently incorporated into zooplankton lipids. Diatom markers were also prominent in the lipids of pre-bloom and bloom phytoplankton at Sunnyside; post-bloom phytoplankton showed higher proportions of 18:0, 18:1o}9, and 18:40}3, characteristic and often major fatty acids of dinoflagellates. The fatty acids of the digestive gland of P. magellanicus reflected the fatty acid composition of the phytoplankton. whereas those of the adductor muscle were practically unaffected by the composition of the food. This organ- specific response of an animal to the fatty acid composition of the diet is examined in terms of different applications of the fatty acid marker concept. © 1997 Elsevier Science Ltd

In t roduct ion Lipids are a heterogeneous group of molecules involved in many vital functions in aquatic organisms (Sargent, 1976). They are a compact and concentrated form of energy storage for plants and animals, and constitute a source of essential nutrients, vitamins, and chemical messengers. Many studies have now recognized the importance of lipids as biological solvents of hydrophobic pollutants (Harding, 1986; Delbeke et al., 1995). Fatty acid constituents of marine lipids are present in a great structural variety associated with the vast biological diversity of marine life. Most biologically important

¶Corresponding author (Fax: (423) 576-9938).

(Received 20 June 1996; accepted 10 April 1997)

739

740 G.E. NAPOLITANO ETAL.

fatty acids are synthesized de novo in the phytoplankton, and are transferred to zooplankton and other primary consumers (e.g. Neal et al., 1986). Fatty acids are major constituents of every l iving cell, and eventually become the predominant lipids of marine particulate organic matter. Fatty acids are increasingly being used as chemical markers of biogeochemical processes and trophic relationships (e.g. Clarke et al., 1987; Eder- ington eta/. , 1 995; Napolitano, 1998). To be useful as atrophic marker, a fatty acid must be synthesized at low trophic levels and then transferred unchanged (or in a recog- nizable form) to upper levels of the food web. Significant advances in analytical chemistry, particularly in gas-chromatography (GC), have led to the use of fatty acids, sterols and hydrocarbons as important tools in the elucidation of trophic relationships (Sargent and Whittle, 1981; Kharlamenko et al., 1995), and in the detection of sources and sinks of discrete pools of organic matter in sea waters and sediments (Saliot et al., 1991 ).

Potential fatty acid markers include those with peculiar structures, unusual unsa- turation patterns, or odd carbon numbers (Paradis and Ackman, 1977). Some polyunsaturated fatty acids (PUFA), in particular 20:50)3 and 22:6co3, are essential for marine animals, and therefore their synthesis at higher trophic levels is negligible. Since these PUFA are synthesized de novo by marine plants, selected bacteria and are then assimilated and retained by marine animals, they constitute particularly useful trophic markers.

There are a number of studies on lipids and fatty acids of natural zooplankton populations at high latitudes (Kattner et al., 1983; Sargent et al., 1985; Clarke et al., 1987), as well as, in temperate (Moreno et al., 1979) and tropical regions (Morris and Hopkins, 1983). However, most of the literature on the fatty acid composition and metabolism in phytoplankton species is based on studies carried out with mono-algal cultures (see reviews by Kayama and Sato (1989) and Brown et al. (1989)). Although these studies are very important, they are not always closely related to the natural environment. This research reports the fatty acids of marine communities in the Bahia Blanca estuary, in the southern coast of Buenos Aires province (Argentina) and at Sunnyside, Trinity Bay, Newfoundland (Canada). Fatty acid markers were used to investigate trophic relationships between phytoplankton blooms and the major local primary consumers: a mixed zooplanktonic assemblage dominated by the calanoid copepod Acart ia tonsa in Argentina, and a population of the bivalve Placopecten magel lan icus in Newfoundland. Two disparate sets of data from studies dealing with food web dynamics are integrated by using the common approach of fatty acids as trophic biochemical markers.

Materials and Methods Study areas and sampling. The Bahia Blanca estuary is located in southeastern Buenos Aires province (Argentina, 38 ° 45'S 61 o 45'W), with an area of approximately 1300 km 2 (Fig. 1 ). It is a relatively shallow, well mixed and turbid body of water. The water temperature fluctuates from 4 to 25°C, with a maximum in Feb- ruary/March and a minimum in July/August. Station 1 was located in Arroyo Maldonado, a creek strongly influenced by tide action. Primary productivity in Bahia Blanca is high, reaching values of up to 300 mg C m-3h -1 (Freije et aL, 1980). The phytoplankton of Bahia Blanca is dominated by diatoms, espe- cially during the development of an unusual winter-spring bloom.

Plankton sampling was carried out at three stations in the Bahia Blanca estuary during 1982. Plankton was sampled at three consecutive stages of the phytoplankton bloom: early-bloom (August), middle-bloom (Sep- tember), and post-bloom (January-February). Phytoplankton was collected in a 30p.m mesh net horizontally hauled for 5 rain at the subsurface, and filtered through a sieve (1001~m screen) to remove large zooplankton. Chlorophyll a was extracted from the seston in 1 I of seawater with 90% acetone and measured spectropho- tometrically according to Strickland and Parsons (1972). Zooplankton was collected with a 2001_Lm net, horizontally and vertically hauled for 8 rain. An aliquot of each sample was fixed in 4% formaldehyde neutralized

FATTY ACIDS AS TROPHIC MARKERS OF PHYTOPLANKTON BLOOMS 741

62 ° 25 ' 62 = 20 ' 62 ° 15 q 62 ~ 10'

FIG. 1. STUDY AREA SHOWING THE LOCATION OF THE SAMPLING STATIONS IN THE BAHIA BLANCA ESTUARY (BUENOS AIR ES, ARG ENTINA).

with 1% borax and used for microscopic studies. The remaining portion of the sample was used for fatty acic~ analysis.

Sunnyside (47 ° 51 'N, 53 ° 55'W) is located in the northern part of Bull Arm on the east side of the Isthmus of Avalon, Newfoundland (Fig. 2). Bull Arm is a north-south fjord-like body of water 6km in length with ar~ average depth of 300 m. Water depths in the Sunnyside collection area rarely exceed 40m. Mean water temperature at Sunnyside is approximately 6°C with minimum values of -1.5°C between February and April and maximum values of 15°C during September and October. Chlorophyll a concentrations reach maximum levels of about 6 I~g 1-1 during the spring bloom in April/May, and with the exception of periodic increases to about 1.5 I~g I-1 during autumn, concentrations typically range from 0.5 to 1.0 I~g 1-1 throughout the remainder of the year (MacDonald and Thompson, 1985). Water samples from Sunnyside were collected in 1990 at 10 rn depth with a Niskin bottle, before (April), during (May), and after (June) the phytoplankton bloom. Phyto- plankton was concentrated by filtration of the water samples using solvent rinsed Whatman G FC filters. Mature female sea scallops (Placopecten magellanicus), a major suspension feeding bivalve in the area, were collected from a depth of 10 m by SCUBA divers.

Simultaneous analyses of phytoplankton species composition and chlorophyll a concentrations for Trinity Bay are not available. Nevertheless, the occurrence, timing and intensity of the spring phytoplankton bloom of Trinity Bay are documented in a previous study carried out at Sunnyside (MacDonald and Thompson, 1986).

Fatty acid analysis. Plankton from the Bahia Blanca estuary was saponified with 10% KOH in ethanol for 45 rain at 80°C under nitrogen (Christie, 1982). The unsaponifiable lipids were extracted with petroleum ether (b.p. fraction 60-70°C) and discarded. The aqueous solutions were acidified with HCI and the free fatty acids were extracted with petroleum ether and converted to methyl esters (FAME) using 7% BF3 in methanol, according to the method of Morrison and Smith (1964). FAME were analyzed by GC in a column packed with 10% SP-2330 on Chromosorb. Temperature was programmed for a linear increase of 3°C min-1 from 140 to 220°C. Fatty acids were identified by comparison of the retention times with samples of known composition

742 G. E NAPOLITANO ETAL.

C

,,,t c>

J

4 8 ° 00"

47 ° 30' -

47 ° 00'-

54o00 ' 53000 '

i i i

54030 ' 53o30 '

FIG. 2. STUDY AREA SHOWING THE LOCATION OF SUNNYSIDE (SS) IN TRINITY BAY (NEWFOUNDLAND, CANADA).

and commercially available standards. The fatty acid chain length was confirmed by catalytic hydrogenation and re-chromatography of the saturated FAME. The number of double bonds was confirmed by separation of the FAME in AgNO3-thin-layer chromatography fol lowed by GC of the fractions.

Lipids from phytoplankton and sea scallops from Sunnyside were extracted with 1:2 v lv chloroform- methanol (Bligh and Dyer, 1959). FAME were obtained by treatment of the extract with 10% BF3 in methanol (Morrison and Smith, 1 964). Analysis of sea scallop and phytoplankton FAME were carried out on a Perkin- Elmer Model 8420 GC (Perkin-Elmer, Norwalk, Connecticut) equipped with a bonded polyglycol (SUPEL- COWAX-10) flexible fused silica capillary column (30m in length x0.25 mm i.d., Supelco, Inc., Bellefonte, Pennsylvania), as described earlier (Napolitano et aL, 1992).

The fatty acid nomenclature used here is of the form 18:2co6, where "18" designates the total number of carbon atoms, "2" the number of cis double bonds, and "co6" the position of the first double bond from the methyl end (co) of the molecule.

FATTY A C I D S A S T R O P H I C M A R K E R S OF P H Y T O P L A N K T O N B L O O M S 743

R e s u l t s a n d D i s c u s s i o n Chlorophyll a concentrations and species composition of the plankton in the Bahia Blanca estuary Figures 3 and 4 show the seasonal variation of chlorophyll a concentrations in stations 1 and 2, respectively, in the Bahia Blanca estuary. Chlorophyll a concentrations were low or moderate (1-20p.g1-1) during summer and fall, and increased sharply (up to 40 I~g 1-1 ) as a result of the winter-spring algal bloom, which usually starts at the end of June and last until October. The concentration of chlorophyll a reached a maximum value (~4011g 1-1) in mid-September during 1 982 and at the beginning of September during the following year.

The taxonomic compositions of phytoplankton and zooplankton for all the stations and sampling times at the Bahia Blanca estuary are presented in Table 1. The seston of the Bahia Blanca estuary mainly consisted of phytoplankton, although some samples included significant amounts of detritus and terrestrial plant debris. The genus Thalas- siosira is the most conspicuous component of the phytoplankton in the area and T. cur- viseriata is the dominant species during the bloom (Gayoso, 1981 ). Other Thalassiosira species present in the bloom were T. anguste-lineata, T. pacifica, T. rotula and T. hiber- nalis. Ditylum brightwelli was also a predominant species during the bloom period at all the stations. Zooplankton in Bahia Blanca estuary was dominated by the calanoid copepod Acartia tonsa. Other zooplankters were: harpacticoid copepod Eutarpina acuti- frons, decapod Peisos petrunkievichi, chaetognath Sagitta sp. and cirripede nauplii.

Fatty acid composition of the phytoplankton and zooplankton in the Bahia Blanca estuary Table 2 presents the fatty acid composition of the lipids in the phytoplankton and

zooplankton of Bahia Blanca during the 1982 algal bloom. The major saturated fatty acids (SFA) of the phytoplankton were 14:0, 16:0, and 18:0, the monounsaturated (MUFA) 16:1007 and 18:1co9, and the polyunsaturated (PUFA) 20:5co3 and 22:6co3

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50

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FIG. 4. SEASONAL VARIATION OF THE CONCENTRATION OF CHLOROPHYLL a IN STATION 2 OF THE BAHIA BLANCA ESTUARY DURING TWO CONSECUTIVE YEARS.

(Table 2). Other important components were 16:2, 1 6:3, 1 8:2c06, and 1 8:3c03. Palmitic acid (16:0) is qualitatively one of the most important fatty acids in algae as well as in animals. In general, it is found in diatoms in smaller amounts than 16:10)7 (Brown eta/. , 1989; Kayama and Sato, 1 989), in contrast to the amounts in other algae and in zooplankton. Figure 5 shows variation in the eleven quantitatively most important fatty acids during the three phases of the algal bloom. If the 16:1c07/16:0 ratios are _> 1.0, then the fatty acid composition reflects a dominance of diatoms (Napolitano eta/., 1 990; Napolitano and Ackman, 1 992), which was observed for the phytoplankton during late winter and spring at stations 1 and 2. The 1 6:1{~7/1 6:0 ratio at station 3 differed from the other sites, having a value <1 for the winter sample. The low 16:1o)7/16:0 ratio suggested a delayed development of the phytoplankton bloom in the outer reaches of the estuary. Observations over a number of years have shown that chl a concentrations and phytoplankton counts at the external reaches of the estuary are lower, and maximum values occur later than in the inner reaches (Gayoso, unpublished results). The major 18:1 isomers (i.e. 18:1c09 and 18:1c07) are scarce in diatoms (Brown et a/., 1989; Kayama and Sato, 1989); 18:1~9 was found at very high concentrations (i.e. 17.4% of the total fatty acids) in the spring samples at station 3. Under the GC condi- tions, 1 8:1 may elute with one or more 16:3 isomers, which have been reported at high concentrations in diatoms (e.g. Ackman et al., 1968; Napolitano et al., 1 990). However, the identification of 18:1 was confirmed by AgNO3-TLC and GC of the resulting bands. In addition, the spring samples at station 3 showed amounts of saturated and monounsaturated fatty acids expected in samples with a significant amount of detritus, since these fatty acids are the most resistant to photo-oxidation processes occurring in the upper waters (Armstrong et al., 1966). A 16:1 ~07/1 6:0 ratio < 1 in the phytoplankton of post-bloom samples at all the stations is related to the presence of relatively low algal biomass (as expressed by low chlorophyll a concentrations, Figs 3 and 4) and relatively high proportions of detritus and micro-zooplankton.

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746 G. E. NAPOLITANO ETAL.

TABLE 2. FATTY ACID COMPOSITION OF TOTAL LIPIDS IN PHYTOPLANKTON

AND ZOOPLANKTON IN BLANCA ESTUARY (SAMPLING STATION 1 ) DURING

THE SPRING BLOOM (PERCENTAGE OF TOTAL FATTY ACIDS OF DUPLICATE

SAMPLES ± sd, n=2

Fatty acid Phytoplankton Zooplankton

14:0 12 7 :L0.1 4.1 i 0 . 2

15:0 1 1 {- 01 0.8 -50.05

16:0 1 7 5 ± 0 2 24.1 ± 1 2

16:1m7 23 5 ± 0 2 6.6 ±0.05

16:2 1 2 ± 0.01 1.5 ± 0.2

16:3 4 4 ± 0.05 1.5 ± 0.2

16:4 31 ±0.05 tr 18:0 42±0 .01 16 .2±22

18:1 ~,~9 4.5 ± 0.01 25.0 ± 33

18:2~,~6 1.3 ± 0.04 1.9 ± 0.2

18:3c,~3 1 0 ~0.01 1 7 ± 0 7

18:4~o3 2.8 ± 01 1.0±001

20:1 nd tr

2 0 : ~ 6 1 9 ± 0.02 1.0 ± 01

20:5e~3 98 :t- 0.2 6.2 ± 25

22:1 nd tr

22:3 0 1 tr 22:4 2.05 ± 0.05 tr 22:&,~3 -: 01 tr

22:6{~3 6.6 ± 0.3 5.3 ± 2.3

SFA 35.5±0.4 4 5 . 2 ± 3 7

MUFA 20.0 ± 0.2 31.6 ± 3.3

PUFA 3 4 2 ± 0 . 8 201 ±6.1

An unusual feature of the fatty acid composition of the samples collected in station 2 of the Bahia Blanca estuary was the higher concentration of linoleic acid (18:20)6), which comprised 8.7% of the total fatty acids during the post-bloom period. The proportion of 18:2o)6 in the phytoplankton of the other sampling stations of the Bahia Blanca estuary (Fig. 5) and in other marine coastal seston (e.g. Parrish et a/., 1996) varied between 2 and 4% of the total fatty acids. The concentration of 18:20)6 found in the post-bloom samples at station 2 was significantly higher (p<0.05 , t-test) than in samples taken during the early and mid-stages of the algal bloom (Fig. 5). Linoleic acid is a minor fatty acid in diatoms and is not common in zooplankton, but it may be an important fatty acid in the lipids of green algae (Napolitano, 1997). However, green algae or the presence of high concentrations of other green alga fatty acid biomarkers (such as 18:30)3) were not detected in these samples. In previous years, the concentration of 1 8:20)6 in the lipids of the particulate organic matter (size range 100-200 #m) at station 2 was measured up to 1 7% of the total fatty acids (Pollero, unpublished data). In all of these cases, macroscopic examination of the samples revealed the presence of plant debris, including seed fragments. The large proportion of 18:20)6 is attributed to agricultural products, in which this fatty acid is a major component of seed lipids. Seeds (wheat and other crops) are routinely scattered into the bay when loading large grain shipments from Ingeniero White Harbor (Fig. 1 ). Linoleic acid performs several metabolic functions and is an essential dietary component for animals. The large quantity of 18:20)6 in the particulate matter in the Bahia Blanca estuary could be of trophic rele- vance since it is utilized by the zooplankton as well as by other components of the food web in this area (see below).


B 1 0

Station 1

o

"~ 20

m 1 "6 =o o - i

Station 2

Station 3

o 20 F

k o 0 / •

Fatty Acids

FIG. 5. FATTY ACID COMPOSITION OF THE PHYTOPLANKTON FROM THE BAHIA BLANCA ESTUARY DURING DIFFERENT PHASES OF THE SEASONAL ALGAL BLOOM.

Despite the dramatic changes in the phytoplankton biomass associated with the development of the phytoplankton bloom (Figs 3 and 4), the bulk of the fatty acid composition of the phytoplankton of the Bahia Blanca estuary showed relatively little variation. One of the few consistent observations was a significant increase (p < 0.05) in the concentration of 20:5(o3 in the lipids of bloom phytoplankton at station 3 as compared with their early- and post-bloom counterparts (Fig. 5). Stations 1 and 3 also exhibited maximum concentrations of =-Iinolenic acid (18:3o)3) during the phytoplankton bloom. =-Linolenic acid is an important structural component of chloroplast glycolipids (Douce et a/., 1990), and its concentration is expected to be higher in algal


populations undertaking rapid cell divisions. Laboratory and enclosure studies have demonstrated that the log-growth phase of phytoplankton is associated with a high content of 18:3c~3 (e.g. Piorreck et aL, 1984), and that senescent stages of algal cultures are characterized by a relatively low proportion of o)3-PUFA, including 18:3c~3 (Piorreck et al., 1984; Fraser et al., 1989).

The dominant fatty acids in the lipids of the zooplankton from the Bahia Blanca estuary were the SFA 16:0 and 18:0, the MUFA 16:1co7 and 18:1c09, and the PUFA 22:6c~3 and 20:5ce3 (Table 2). In contrast with the relative stability of the fatty acid composition of phytoplankton during the development of the seasonal bloom, the lipids of the zooplankton presented consistent and pronounced trends clearly observable at the three stations (Fig. 6). Total SFA, in particular 16:0 and 18:0, decreased significantly (p < 0.05) from the early-bloom to the post-bloom period. Other important fatty acids, such as 18:1ce9 and 18:2co6 also showed relatively low concentrations in the post- bloom samples (except in station 2). The zooplankton in the Bahia Blanca estuary was also characterized by very low concentrations of 20:1 and 22:1 isomers (Table 2). The MUFA 20:1co9 and 22:1col 1 are particularly abundant in calanoid copepods that have wax esters as their main storage lipid (Sargent, 1976; Clarke et al., 1 987). Thus, from the fatty acid composition of the zooplankton of the Bahia Blanca estuary it can be inferred that the calanoid copepod A. tonsa, the dominant species in the zooplankton of most of the samples analyzed, does not store significant amounts of wax esters. The lack of appreciable amounts of wax esters in an herbivorous calanoid copepod as A. tonsa is very unusual (Sargent and Falk-Petersen, 1988), and is consistent with the findings of Ederington et al. (1995).

The concentrations of the two major PUFA in the zooplankton at the Bahia Blanca estuary (20:5co3 and 22:6co3) showed a progressive increase (p < 0.05) from August to January, coinciding with the development of the phytoplankton bloom. Changes in the fatty acid composition of the zooplankton clearly reflected the incorporation of lipids of dietary origin, and their desaturation-elongation products (Moreno et al., 1979). A case in point is linoleic acid (18:2ce6), which was present at relatively high concentrations in the particulate organic matter in station 2 (Fig. 5), and appears to be assimilated and incorporated into the lipids of the zooplankton (Fig. 6). The enrichment of 18:2c06 in the lipids of the zooplankton was particularly noticeable during the early- and post-bloom periods, when the input of phytoplankton derived fatty acids was relatively less important. This represented a singular case of incorporation of terrigenous organic material into a marine food web.

Fatty acid composit ion of phytoplankton at Sunnyside The lipids of the phytoplankton collected at Sunnyside during the spring bloom con-

tained approximately equal proportions of SFA, MUFA, and PUFA (Table 3). The spring phytoplankton also showed a fatty acid profile consistent with the dominance of diatoms, as indicated by a 1 6:1co7/16:0 ratio close to 1.0, high levels of C16-PUFA (9.2%, including 16:4cel), and 20:5c~3 (12.1%). The main fatty acids in the phytoplankton before, during and after the spring bloom were the SFA 14:0, 16:0, and 18:0; MUFA 16:1ce7, 18:1ce9, and the PUFA 20:5ce3 (Fig. 7). Compared with the phytoplankton of the bloom stage, the pre- and post-bloom phytoplankton contained relatively less PUFA and relatively more SFA. Pre-bloom and bloom phytoplankton had similar proportions of 16:0, 16:1 co7, and 20:5c03, indicating that diatoms were important


3°it Station 1

- - 1 0

Station 2 3O

"~ 20

s0 Station 3

Fatty Acids FIG 6. FATTY ACID COMPOSITION OF THE ZOOPLANKTON FROM THE BAHIA BLANCA ESTUARY DURING DIFFERENT PHASES OF THE SEASONAL ALGAL BLOOM.

in both cases. Post-bloom phytoplankton, on the other hand, showed considerably lower levels of 1 6:1o)7, 1 6:4o)1, and 20:50)3, and higher concentrations of 16:0, 1 8:0, 18:1(o9, and 1 8:40)3. These changes in fatty acid composition indicated a decrease in the importance of diatoms as contributors to the phytoplankton biomass, and an increase in phytoflagellates and detritus as constituents of the particulate organic matter in post-bloom situations.

High concentrations of (03-PUFA and low concentrations of SFA were also observed in the lipids of the spring phytoplankton from the Bahia Blanca estuary. These lipids are commonly found in phytoplankton during this seasonal bloom phase and are characteristic of the lipids of natural and cultured algae undergoing rapid cell division (Piorreck

750 G. E. NAPOLITANO ETAL.

TABLE 3. FATTY ACID COMPOSITIONS OF PHYTOPLANKTON (MEAN OF TWO REPLICATES), SEA

SCALLOP ADDUCTOR AND DIGESTIVE GLAND FROM SUNNYSlDE DURING THE SPRING ALGAL BLOOM (PERCENTAGE OF TOTAL FATTY ACIDS, MEAN ±sd, n=3)

Phytoplankton A. muscle D. gland

12:0 1.1 nd 0.09 ± 0.01 14:0 10.7 1 . 8 ± 0 2 3.7±0.3

TMTD nd nd 2.3±0.3 15:0 1.3 0.86 ± 0.02 0.29 :E 0.08

16:0 20.2 19.1 :L0.2 125±0 .9

16:1{,)7 20.0 1 9 ± 0 2 10.9~ 1.1 16:1 ~L~5 0.75 0.47 ± 0.31 ~ 0.02

C16-PUFA 5.8 2.5 ± 0.2 4.0 ± 0.3 16:4~'~'4 3.4 1 4 ± 01 5.0 ± 0.2

18:0 3.7 42 ± 0.9 2.0 ± 0.3 18:1c09 45 3.2± 1.5 3.3 ± 0.1

18:1 c,~7 22 4.9 ± 0 7 5.8 ± 0.5 18:1 ~,~5 0.5 0.32 ± 0.03 0.33 ± 0.1 18:2co6 1 1 0.8 ± 0.03 1.5 ± 0.4

18:3~,~3 < 0.1 0.96 ± 0.05 1.7 ± 0.2 18:4~'t',3 2.6 3.8 + 0.3 7.9 ± 0.9

20:1 {,~11 nd 0.86 ± 0.4 0.58 ± 0.5 20:1 {,~9 nd 0.84 ± 0.3 0.77 ± 0.02 20:1 t~7 nd 0.43 ± 0.3 0.69 ± 0.08

20:2NMIDa nd 024 ± 0.2 0.14 ± 0.07 20:2NMIDb nd 014 ± 0.1 0.15 i 0.01

20:2~,)6 0.7 1.5 ± 1.1 0.43 ± 0.05 20:41:~6 03 1 9 ± 0.1 0.70 ± 0.05

20:4~:,~3 0.3 077 ± 0.1 0.96 ± 009 20:5{~3 121 18.8 ± 1.3 21.8 ± 04 22:5(o6 nd 0.57 ± 0.06 2.6 ± 0.3 22:5~,~3 2.2 1 1 ± 0.2 0.87 ± 0.3 22:6~3 4.8 23.7 ± 0.9 54 ± 0.8

SFA 37.0 25.9 20.5

MUFA 31.1 14.3 22.7 PUFA 33.3 58.2 53.1

nd = not detected, tr = trace amounts

35

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F a t t y A c i d s

FIG 7. FATTY ACID COMPOSITION OF THE PHYTOPLANKTON FROM SUNNYSIDE DURING DIFFERENT PHASES OF THE SEASONAL ALGAL BLOOM (DUPLICATE SAMPLES).


et al., 1984; Fraser et al., 1989). Phytoplankton cells in log-phase allocate large portions of photosynthesized carbon to the formation of structural lipids, typically composed of unsaturated fatty acids (Sargent et al., 1985).

Fatty acid composition of the adductor muscle and the digestive gland of sea scallops from Sunnyside

The fatty acid composition of P. magellanicus has recently been studied (Napolitano et al., 1992; Napolitano and Ackman, 1993), and other scallop species have also been investigated (see reviews by Joseph (1982, 1989)). However, most of the earlier information on pectinid fatty acids refers exclusively to the adductor muscle (Gruger et al., 1964; Stansby and Hall, 1967; Exler and Weihrauch, 1977). These past reports showed reasonable agreement with the fatty acid composition of the major components described here. The lipids of the scallop adductor muscle were composed of approximately 25% SFA, 1 5% MUFA, and 60% PUFA (Table 3). The principal fatty acids were 16:0, 18:0, 18:10)7, 18:4(o3, 20:50)3, and 22:60)3. This fatty acid composition showed a remarkable consistency, changing little during the different phases of the bloom (Fig. 8), apart from a decrease in the concentration of 16:0 from 18.7 and 19.1%, before and during the algal bloom, respectively, to 12.9% after the bloom.

The lipids of the digestive gland were composed of approximately 25% SFA, 25% MUFA, and 50% PUFA (Table 3). Predominant fatty acids in this organ were 14:0, 16:0, 16:10)7, TMTD (4,8,12-trimethyltridecanoic), 16:4(01,18:10)9, 18:10)7, 18:3(o3, 20:50)3, and 22:60)3. In contrast with the stability of the fatty acid composition of the adductor muscle, the fatty acids of the digestive gland exhibited a series of compositional shifts, reflecting the evolution of the algal bloom (Fig. 9). The main changes observed were increments (p<0.05) in the proportion of 18:10)9, 18:40)3, and 22:60)3, before the bloom, and of 16:10)7, 16:40)1, and principally 20:50)3 during and after the bloom. The fatty acids characteristic of the pre-bloom period (i.e. 18:1(o9, 18:40)3, and 22:60)3) are

30

2 5 ffl "U

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~ 10 0

5

~3

Fatty Acids

FIG. 8. FATTY ACID COMPOSITION OF THE ADDUCTOR MUSCLE OF THE SEA SCALLOP P. magellanicus FROM SUNNYSIDE DURING DIFFERENT PHASES OF THE SEASONAL ALGAL BLOOM.

752 G. E, NAPOLITANO ETAL.

£

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~ Pte-bloom 25 - B~m Post~loom

20

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Fatty Acids

FIG. 9, FATTY ACID COMPOSITION OF THE DIGESTIVE GLAND OF THE SEA SCALLOP P. magellanicus FROM SUNNYSIDE DURING DIFFERENT PHASES OF THE SEASONAL ALGAL BLOOM.

common and often major constituents of dinoflagellate and prymnesiophyta lipids (Ackman et al., 1968; Volkman et al., 1981 ; Napolitano et al., 1990, 1995). On the other hand, those fatty acids exhibiting elevated concentrations during bloom and post-bloom periods (i.e. 16:1 e)7, 16:1o)4, and 20:5co3), are well established biochemical markers for diatoms (e.g. Volkman et al., 1989; Napolitano, 1994; Napolitano et al., 1994; Kharla- menko et al., 1995; Parrish et al., 1996).

Organ-specific fatty acid compositions in animals always consist of arrangements of the same fatty acids present in different proportions. The distribution of TMTD between tissues in P. magel lanicus is a special case, since this fatty acid was present in the digestive gland (c. 2.3-4.4% of the total fatty acids), but was not detected in the adductor muscle. TMTD is derived from the breakdown of chlorophyll a (Hansen, 1980) and is stored in the digestive gland, but apparently is not assimilated or transported to other organs. A more detailed study of the anatomical distribution of fatty acids in P. magel lanicus (Napolitano and Ackman, 1993) showed that TMTD was undetectable (or present only in trace amounts) in male and female gonads, gills, and mantle. This highly restricted anatomical distribution of TMTD was not observed in the organs of the bay scallop Argopecten irradians, nor in the freshwater snail Elimia claveiformis, where the fatty acid showed a widespread distribution in all the major organs (Napolitano, unpublished data).

The lipids of the adductor muscle of the sea scallop are mainly composed of structural phospholipids, sterols, and trace amounts of triacylglycerols, whereas the digestive gland lipids are about 60% triacylglycerols (Napolitano and Ackman, 1992). As a con- sequence of these different lipid class compositions, the fatty acid data from these two organs provide distinct but complementary information. The fatty acid composition of the triacylglycerols and triacylglycerol-rich animal tissues is species-specific but reflects, to some extent, the fatty acid composition of the diet. Furthermore, the digestive gland contains approximately 35% phospholipids, but in contrast with the adductor muscle, a considerable part of these phospholipids are food particles at different stages of the


digestive process (Chang et al., 1989), and not from the animal's structural lipids. Therefore, both tr iacylglycerol and phospholipid fatty acids of the digestive gland reflect the fatty acid composit ion of the food and are particularly suitable for ecological studies of food qual i ty and trophic relationships.

In contrast, the fatty acid composit ion of phospholipids in the scallop adductor muscle (or the l ipids of other non-fat ty tissues) are primarily determined by cellular membrane funct ions and environmental condit ions other than diet. It has been shown that the gonad and the digestive gland of sea scallops incorporate dietary fatty acids and sterols during a six week laboratory feeding experiment, whi le the adductor muscle maintains the original l ipid composit ion, independent of the diet (Napol i tano et al., 1993). The results presented here are clearly consistent with these previous experi- ments, showing that the fatty acid composit ion of the adductor muscle is not substan- t ial ly affected by the phytoplankton bloom. The relative independence of the lipid composit ion of an animal tissue from the diet opens the opportunity for studies of thermal adaptation (Roy et al., 1 992; Staurnes et al., 1994), and even to more sophisti- cated analysis of population structure, based on phospholipid fatty acid compositions, as described by GrahI-Nielsen and Mjaavatten (1992).

Acknowledgements-We thank Prof. R. G. Ackman (Technical University of Nova Scotia) and A. J. Stewart (ORNL) for their support. We also thank J. E. Richmond (ORNL) for technical assistance. G. E. Napolitano was supported by the Consejo Nacional de Investigaciones Cientificas y T~cnicas (CONICET, Argentina) and by fellowships from the faculty of Graduate Studies of Dalhousie University. Financial assistance from Natural Sciences Research and Engineering Council of Canada operating grants to Drs R. J. Thompson, B. A. Mac- Donald and R. G. Ackman is appreciated. C. C. Parrish (Memorial University of Newfoundland) provided constructive comments on a previous version of this manuscript. Writing of this report was supported in part by an appointment of G.E.N. to the Oak Ridge National Laboratory Research Associates Program administrated jointly by ORNL and by the Oak Ridge Institute for Science and Education. Oak Ridge National Laboratory is managed by Lockheed Martin Energy Research Corp. for the U.S. Department of Energy under contract No. DE-ACO5-96OR22464. This is Environmental Sciences Division publication number 4673.

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Fatty acids as trophic markers of phytoplankton blooms in the Bahía Blanca estuary (Buenos Aires, Argentina) and in Trinity Bay (Newfoundland, Canada)