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Distribution of algal chlorophyll and carotenoid pigments in a stratified estuary: the Krka River, Adriatic Sea
Marine Chemistry, 32 ( 1991 ) 285-297 Elsevier Science Publishers B.V., Amsterdam
285
Distribution of algal chlorophyll and carotenoid pigments in a stratified estuary: the Krka River, Adriatic Sea V. Denant t, A. Saliot t,* and R.F.C. M a n t o u r a 2 tLaboratoire de Physique et Chimie Marines de l'Universitb Pierre et Marie Curie, UA CNRS 353, Tour 24-25, 4 Place Jussieu, 75252 Paris Cbdex 05 (France) 2plymouth Marine Laboratory, Prospect Place, West Hoe, Plymouth PL 1 3DH (Gt. Britain) (Received September 28, 1989; revision accepted May 10, 1990)
ABSTRACT Denant, V., Saliot, A. and Mantoura, R.F.C., 1991. Distribution of algal chlorophyll and carotenoid pigments in a stratified estuary: the Krka River, Adriatic Sea. Mar. Chem., 32: 285-297. The detailed distribution of algal chlorophyll and carotenoid pigments was determined around the halocline (freshwater-seawater interface) in the Krka Estuary on the east coast of the Adriatic Sea, in May 1988. After collection of water along the estuary, particulate matter was extracted and analyzed for pigments by high-performance liquid chromatography coupled with absorbance and fluorescence detection. Bottom marine waters were characterized by lower chlorophyll a (chl a) concentrations than encountered in surface waters, decreasing downstream from 0.50/tg 1- t to 0.16/zg 1- t at the marine end-member. The highest concentrations of chl a (up to 26.34 #g 1- ~) were found in the interracial layer, an particularly at one station located off the city of ~ibenik, where high inputs of nutrients supported the accumulation of living algae at the halocline. Fucoxanthin was the most abundant carotenoid, which indicates a euryhaline dominance of diatoms in the estuary, whereas the dinoflageUate-derived carotenoid peridinin was confined to the interfacial and bottom saline waters of the inner estuary. High concentrations of alloxanthin and chl b were found in the interfacial layer, which also suggests an accumulation of Cryptophyceae and green algae in the inner estuary. Phaeophorbides showed higher concentrations in bottom waters than in surface waters, whereas the highest concentrations occurred in the interracial layer. These high levels could reflect a density trapping of dead cells in an early degradation state, as suggested by the importance of allomerized chl a and chlorophyUide a vs. total chl a, or of faecal pellets originating from zooplankton grazing in the interfaciai layer.
INTRODUCTION
The importance of acquiring further knowledge of the biogeochemistry of organic matter in estuarine systems, in particular that at the freshwater-seawater interface, withrespect to the high productivity and the various biogeo*Author to whom correspondence should be addressed.
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chemical processes involved in these areas, has been highlighted by several authors (Sholkovitz, 1976; Morris et al., 1978; Cadee, 1982; Wollast, 1983; Mantoura and Woodward, 1983; Ertel et al., 1986; Hedges et al., 1986; Mantoura, 1987; Zuti6 and Legovir, 1987; Lee and Wakeham, 1988, and references therein; Relexans et al., 1988; Saliot et al., 1988; Denant and Saliot, 1990). The freshwater-seawater interface has been described as a filter which is capable of removing living biomass and detritus; this leads to new biological properties associated with bio-accumulation, bio-degradation and, more generally, bio-transformation processes. These processes could result in selective organic film formation, flocculation and sedimentation (Cuker, 1987: Mantoura, 1987; ~uti6 and Legovir, 1987; Svetlici6 et al., 1991 ). In most tidal estuaries, the difficulty encountered in field observations is mainly due to the very variable stratification of waters according to river flow and tidal coefficient. To study in detail the processes that occur in both fresh and marine waters and in the boundary layer, we have chosen the Krka River, which enters the Adriatic Sea. In this stratified karstic estuary, the suspendedmatter load is very low and the freshwater-seawater boundary is highly compressed and stable, and is easily sampled by divers. To study the evolution and the nature of organic matter in both river and marine waters within the estuary, and the effect of the freshwater-seawater interface on natural organic matter derived from phytoplankton, we have focused on the analysis of algal chlorophyll and carotenoid pigments, using high-performance liquid chromatography (HPLC). These organic markers provide chemotaxonomic and physiological information on phytoplankton populations and, more generally, on the extent of degradation of algal-derived organic matter (Jeffrey, 1974; Johansen et al., 1974; Goodwin, 1976; Liaaen-Jensen, 1977; Mantoura and Llewellyn, 1983; Klein and Sournia, 1987; Vernet and Lorenzen, 1987). In this paper, we will attempt to determine the vertical and axial changes in the nature and concentration of pigments that occur (1) in the surface river waters during mixing with seawater in the estuary; (2) in the bottom marine waters, which enter the estuary for a distance of ~ 25 km; and (3) at the freshwater-seawater interface at three representative stations sampled in spring 1988, characterized by a relatively high phytoplanktonic productivity. MATERIAL AND METHODS
Sampling sites The Krka River is a karstic river entering the Adriatic Sea (Fig. 1 ). The Krka River estuary waters are characterized by a low suspension load and low terrigenous inputs; they are poor in nutrients ( ~ v a n i ~ and Gl~etir, 1986 ), with the exception of high phosphate near the harbour area of~ibenik. In this
287
ALGAL CHLOROPHYLL AND CAROTENOID PIGMENTS IN THE KRKA
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Fig. 1. Krka Estuary, May 1988 cruise: location of sampling sites and distribution of salinity along the estuary. stratified estuary, the boundary layer, which is easily visible and sampled by divers, is always situated in the euphotic zone. Water samples were collected in the Krka Estuary in May 1988 throughout the estuary, from the waterfalls at the head of the estuary to marine waters off Zlarin island. Stations were sampled for surface waters (freshwater at Eo, with an increasing salinity up to the marine point, M.P.; Fig. 1 ), deep marine waters and the boundary layer at stations E2, E3 and E4a (the last station was located off the city of ~ibenik).
Collection of samples Water samples (20 1) were taken by in situ pumping and, for the interface, with the help of a diver. Water was filtered in subdued light a few hours after sampling, on glass fibre filters (Whatman G F / F , 0.7-#m pore size, 47-ram diameter). Filters were precleaned by rinsing with acetone for 24 h in a Soxhlet apparatus. Filters were always kept, even during transport, in glass tubes in a freezer ( T < - 20 ° C), and were analyzed within 1 month after the sampling.
Analysis Each filter, still frozen, was extracted with 3 ml of a mixture of cold acetone and HPLC-grade MilliQ water (9: l, v / v ) in subdued light. Acetone extract (500/A) was sampled with a syringe and added to 150/tl of ion-pairing reagent (solution P) prepared from 1.5 g of tetrabutylammonium acetate and
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1 Absorbance
5
10 Time
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Time
( mm
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Fig. 2. Reverse phase H PLC absorbance (2 = 440 nm) and fluorescence ('~excitation = 440 nm: 2emi.ion = 500--700 nm) chromatograms of carotenoids and chlorophyll pigments from an extract of particles collected at station E 3. at 10-m depth. D.V. = dead volume. Peak identities: ( I ) chlorophyll c: (2) peridinin; (3) fucoxanthin-like compound No. 1; (4) fucoxanthin; ( 5 ) fucoxanthin-like compound No. 2; (6) diadinoxanthin; (7) alloxanthin; (8) zeaxanthin/lutein; (9) chlorophyll b; (10) allomerized chlorophyll a; ( 11 ) chlorophyll a; ( 12 ) ~-carotene; (13) phaeophorbide a-like compound; (14) phaeophorbide a.
7.7 g of ammonium acetate made up to 100 ml with water. After thorough mixing, 100/zl were injected into the chromatograph, using a Rlaeodyne model 7125 injector valve. Chromatographic separation was performed following a methodslightly modified from that of Mantoura and Llewellyn ( 1983 ). Separation of pigments was obtained using a 25-cm X 1/4-in. column packed with 5-/1m silica (C18 CDS2, Spherisorb) by linear gradient elution from 100% A to 99% B in 10 min, followed by an isocratic step at 99% B for 12 rain. Solution A was made up of a methanol-water-solutio n P mixture (8" 1 : 1, v / v / v ) . Solution B was made up of methanol-acetone (7: 3, v/v). The HPLC equipment consisted of L.D.C. Milton Roy constametric I and IIt G pumps, a programmable gradient elution system, an L,D.C. Milton Roy Fluoromonitor III fluorescence detector ()~excitation'-- 440 rim; 2em,,io, = 500-700 rim) and
ALGAL CHLOROPHYLL AND CAROTENOID PIGMENTS IN THE KRKA
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a Beckman 165 UV detector used at 2 = 440 nm. Typical absorbance and fluorescence chromatograms of pigments extracted from particulate matter sampled at station E3 under the boundary at 10-m depth are shown in Fig. 2. The pigments were measured using standards (Mantoura and Llewellyn, 1983 ). Within the conditions used, the following remarks should be made: ( 1 ) two phaeophorbides were characterized, and denoted as phaeophorbide a (peak 14) and phaeophorbide a-like compound (peak 13 ), according to the terminology used by BurkiU et al. ( 1987); (2) two compounds eluting very close to fucoxanthin were separated, and were denoted as fucoxanthin-like compound No. 1 (peak 3) and fucoxanthin-like compound No. 2 (peak 5 ).
Summary of pigment distribution interpretation Chlorophyll a (chl a) is a ubiquitous pigment and can be used as a global algal (freshwater and marine) biomass indicator. Different degradation products from chl a will be considered here. Allomerized chl a, chlorophyllide a, have been found after cell lysis by bacteria (Gieskes et al., 1978 ). Chlorophyllide a is generally considered to be an autolytic degradation product of chl a which is reversibly catalyzed by the enzyme chlorophyllase (Bogorad, 1976); thus, it could indicate the presence of senescent cells (Jeffrey, 1974), although it has been also reported to occur during periods of active growth (Ridout and Morris, 1985 ). The phaeophorbides a are also products of degradation of chl a; phaeophorbide a has been often found in faecal pellets (Jeffrey, 1974). Thus zooplankton grazing is considered to be the major source of this pigment (Welschmeyer and Lorenzen, 1985 ). Fucoxanthin and chl c are indicators of diatoms (Goodwin, 1976 ), whereas peridinin and chl c are synthesized by dinoflagellates (Johansen et al., 1974). Chlorophyll b (chl b) has been commonly ascribed to green algae (Jeffrey, 1974). RESULTS
Characteristics of surface waters Chlorophyll a Figure 3 shows the distribution of chl a concentrations along the estuary. Chl a concentrations of surface waters increase from < 1 #g 1- ~ at Eo (pure river water) and E2 (S< 30/00), at the head of the estuary, and reach a maxim u m of 2.05/zg 1-~ at station E4a (S=7%o) near ~ibenik. Thereafter, chl a decreases to 1.08/zg 1- ~at station C2 ( S = 25%o) and 0.38/tg l - ~at the marine end-member (S~ 370/00).
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Fig. 3. Krka Estuary, May 1988: distribution ofchl a concentrations expressed in #g 1- l in surface water, boundary layer and bottom water. Chl a concentration at station E4, boundary layer is out of scale (26.34/tg l - ] ). 0.6-
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Fig. 4. Krka Estuary, May 1988: distributions of allomerized chl a, chlorophyllide a, phaeophorbide a and phaeophorbide a-like compound in surface water, boundary layer and bottom water.
Degradation productsfrom chlorophyllpigments Distributions o f allomerized chl a and chlorophyllide a are very similar to that o f chl a (Fig. 4). Their concentrations are low in the variation ranges 0.01-0.06/tg l - ' and 0.00-0.08/tg 1-1, respectively. These two degradation products represent < 5% o f total chl a.
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ALGAL CHLOROPHYLL AND CAROTENOID PIGMENTS IN THE KRKA
Neither phaeophorbide a nor phaeophorbide a-like compound has been detected in the low-salinity section of the estuary ( S < 3%0). With the increasing influence of marine waters, these two compounds appear, and they reach their highest concentration at the marine point, where they represent up to 16 and 34% of chl a, respectively (Fig. 4).
Taxonomic pigments Fucoxanthin is the predominant carotenoid identified for all samples. Its concentration varies from 0.07 to 0.32/zg 1-1; the minimum value corresponds to the marine reference (Fig. 5 ). Peridinin was not detected in any of the surface samples. The distributions of alloxanthin and chl b are close to that ofchl a. The highest concentrations occur at station E4a - - 0.27 and 0.14 /zg l- 1, respectively.
Characteristics of bottom (marine) waters Chlorophyll a Water samples below the halocline/thermocline are characterized by lower chl a concentrations than in surface waters; the concentration decreases
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Fig. 5. Krka Estuary, May 1988: distributions of concentrations of taxonomic pigments, fucoxanthin, peridinin, alloxanthin and chlorophyll b in surface water, boundary layer and bottom water. (Note the change of scale used for pigment concentration ( 0 - 6 pg l - 1) for the E4a interface sample. )
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V. D E N A N T ET AL.
downstream, from 0.50/~g 1-J at station Eo to 0.16 ~tg 1-J at the marine reference (Fig. 3).
Degradation products from chlorophyllpigments Allomerized chl a has very low concentrations which vary in a narrow range (0.00-0.01/~g 1-J ), whereas chlorophyllide a was not detectable (Fig. 4 ). Phaeophorbides a distributions are most interesting. Phaeophorbide a is m a x i m u m at station E3 (0.27/tg 1- J ) but has a much lower concentration variation (in the range 0.00-0.01/~g 1-j ) for other samples, Phaeophorbide a-like c o m p o u n d has two concentration maxima, which occur at stations E 3 and C2.
Taxonomic pigments Fucoxanthin is the predominant carotenoid, but at a lower level than observed in surface waters (Fig. 5 ). Peridinin is present only at stations E2 and E3. Alloxanthin and chl b are present at low concentrations, which decrease downstream. Two pigments that have a structure close to fucoxanthin which are absent in surface waters are detected here: fucoxanthin-like c o m p o u n d No. 1, present only in samples where peridinin occurs, and fucoxanthin-like c o m p o u n d No. 2 at stations E2, E3, C2 and the marine reference point, where phaeophorbide a-like c o m p o u n d was also detected.
Characteristics of the freshwater-marine water interface Chlorophyll a Chl a concentrations are higher than those observed in surface waters (Fig. 3 ). A very high value of 26.34/tg 1-J was observed at station E4a.
Degradation products from chlorophyllpigments Allomerized chl a and chlorophyllide a show a remarkable increase in terms of absolute concentration (Fig. 4), but not in terms of percentage of total chl a, which has a relative importance of < 2%. Phaeophorbide a-like compound is significantly present at stations E2 and E3, where it accounts for ~ 27% ofchl a.
Taxonomic pigments As for other samples from both surface and bottom layers, fucoxanthin predominates (Fig. 5 ), but here alloxanthin and chlorophyll b are present at relatively high levels, e.g. 1.39 and 0.79/~g 1- J at station E4a. Peridinin and fucoxanthin-like c o m p o u n d No, 1 are present only at station E2, whereas fucoxanthin-like c o m p o u n d No. 2 has a m a x i m u m a t s t a t i o n E3: 0.17 /tg1-1.
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DISCUSSION Considering the three complete vertical profiles obtained at stations E2, E3 and E4a, the freshwater-seawater interface is characterized first by a marked enrichment in chl a. This enrichment has been previously observed during winter conditions in November 1984 (Vili~i~ et al., 1989 ) and in March 1987 (Denant, 1988), and during spring conditions in April 1985 (Vili~i~ et al., 1989 ). Vili~i~ et al. (1989) have documented the vertical distribution ofphytoplankton, and have shown that the selective accumulation of larger phytoplankton cells and the associated high chl a concentrations in the interfacial layer are a consequence of the accumulation of viable freshwater phytoplankton cells in the upper boundary of the interface. Chl a concentrations are highest in the film, and particularly at station E4a, for both winter and spring conditions. These high levels can be easily explained by the proximity to the city of ~ibenik (Fig. l ), as large nutrient inputs, especially of phosphate ( 106 mol year-1; Sekuli~, 1989), are introduced into the estuary from sewage effluents and harbor activities. Distribution profiles of allomerized chl a and chlorophyUide a are close to that of chl a. Their concentrations relative to the parent compound chl a are small, which suggests that, at this time of year, the degradation of organic material of phytoplanktonic origin through senescence of cells and lysis by bacteria is not pronounced. On the other hand, the opposite was observed during winter conditions, when high concentrations of chlorophyllide a occurred at the interface, representing 10-12% of total chl a. However, the possibility of an artefactual formation of chlorophyllide a during the filtration of water samples could not be ruled out (Jeffrey and Hallegraeff, 1987), although the same precautions have been taken during the two cruises. Phaeophytin a was not detected. Two hypotheses can be proposed to explain this: there may be no formation of phaeophytin a in natural conditions, or there may be a rapid transformation into phaeophorbide a. Two phaeophorbides a were detected. Phaeophorbide a-like compound is always predominant over phaeophorbide a, and has a higher concentration in bottom marine waters than in surface waters, whereas the highest concentrations occur in the interfacial layer. Vilici~ et al. ( 1989 ) have observed such an accumulation of dead phytoplankton cells in the interfacial layer. These vertical profiles could be explained by settling followed by density trapping, in the organic film that develops at the interfacial layer, of particles enriched in phytoplankton material, such as faecal pellets that originate from the grazing of phytoplankton by freshwater zooplanktonic organisms. Such particles are known to be enriched in compounds derived from the degradation of chlorophyll and other recently biosynthesized organic material. Phaeophorbide alike compound has been interpreted as an indicator of a more advanced degradation state of chl a (Vernet and Lorenzen, 1987). The value of the ratio
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Phaeo a-like c o m p o u n d / P h a e o a is in the range 1.5-2.2 for surface marine waters. Bottom waters, which are older, as they are transported from the sea into the estuary on a time-scale of the order of a few years (Martin et al., personal communication, 1989 ), are characterized by values of the ratio Phaeo a-like c o m p o u n d / P h a e o a that increase with the residence time of waters: 0.7 at station E 3 and 5 at station E2. Some interesting features appear when we consider the distribution of chemotaxonomically specific pigments. Fucoxanthin is the principal carotenoid pigment, reflecting the wide distribution of diatoms in these waters (D. Vilici6, personal communication, 1989). The abundance of diatoms varies in the following range: 2.67 × 105 cells 1- J at station Eo; between 2.53 × 105 (E2) and 9.4 × 104 (C:) cells 1- ~ for surface waters; and between 5 × 103 (E3) and 5.1 × 104 (C2) cells 1- ~for bottom waters. There is no systematic relationship between the concentration of fucoxanthin and the abundance of diatoms, whereas such a relation was observed during winter conditions. Peridin in was detected only at station E2 and E 3. This restricted distribution of dinoflagellate in estuarine environments was described by Incze and Yentsch in 1981. It was also observed during winter conditions in March 1987 (Denant, 1988 ). Dinoflagellates here develop in interfacial and bottom waters, where the difference of salinity between surface and bottom waters always exceeds 35%0, at the end of the saline front. C o m p o u n d s derived from or having a structure close to fucoxanthin have been previously observed on HPLC and include hexanoyl- and butanoyl-oxyfucoxanthin esters. They are more uniquely synthesized by various species of prymnesiophyte phytoplankton (Gieskes and Kraay, 1983. 1986a, b; Wright and Jeffrey, 1987; Gieskes et al., 1988 ). Two compounds that have a fucoxanthin-type structure have been detected. Fucoxanthin-like c o m p o u n d No. 1 shows an interesting distribution, as it is present only in the samples where peridinin is observed. It is commonly assumed that dinoflagellates do not synthesize simultaneously peridinin and fucoxanthin-derived compounds (Tangen and Bjtirnland, 1981; Wright and Jeffrey, 1987 ). It is probable that the fucoxanthin-like c o m p o u n d No. 1 originates from species that are not dinoflagetlates but that live in close association with them. The fucoxanthin-like c o m p o u n d No. 2 is always found in association with phaeophorbide a-like c o m p o u n d and thus could derive from the degradation of fucoxanthin. High concentrations of alloxanthin and chlorophyll b found in the interracial layer suggest an accumulation of Cryptophyceae and green algae relative to river and marine waters. These two algal classes appear to develop in the estuary between station Eo and Cz, but not in marine waters offthe estuary.
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CONCLUSION
The stratified Krka Estuary could be considered as a unique model for studying the respective impacts of riverine and marine processes on the evolution of phytogenic organic matter. In May 1988, pigment concentrations varied over a large range along the estuary. Chl a concentrations of surface waters largely depend on the mixing between fresh and saline waters. The same tendency is observed for allomerized chl a and chlorophyUide a. Chl a concentrations of marine waters entering the estuary increase from the marine environment to the inner estuary, which suggests a marked influence of surface waters on the productivity of bottom waters, and possible exchange of nutrients across the interface. Other pigment concentrations, such as for fucoxanthin, peridinin, alloxanthin and chl b, reflect the specific distribution of predominant phytoplankton species such as diatoms, dinoflagellates, Cryptophyceae and green algae, respectively. Compounds with a fucoxanthin-type structure show interesting associations: one compound, which correlates with peridinin, suggests the potential presence of phytoplankton species living in close association with dinoflagellates; the other correlates with phaeophorbide a-like compound and thus originates from the degradation of diatom-derived fucoxanthin. The interfacial layer thus appears to be a biogeochemically very active zone for phytogenic organic matter. The highest chl a concentrations occur in the interfacial layer, reflecting the accumulation of living phytoplankton. Accumulation at the boundary is observed for different pigments related to different phytoplankton species, and occurs at different sites of the estuary, depending on the salinity gradient between surface and bottom marine waters: fucoxanthin occurs in high concentration throughout the estuary, as diatoms predominate in both riverine and saline waters. Alloxanthin and chl b are accumulated in the inner estuary, whereas peridinin is found only in a very few bottom and interface samples, reflecting a more patchy distribution of dinoflagellates. The interface appears also to be an efficient trap for dead cells in faecal pellets of zooplankton, as suggested by the relative accumulation of allomerized chl a, chlorophyllide a and phaeophorbides. ACKNOWLEDGEMENTS
We thank Dr. Vera 7.utir, Dr. G. Cauwet and Dr. J.M. Martin, coordinators of the French-Yugoslavian programme on the Krka River, and C. Llewellyn (Plymouth Marine Laboratory ) for training help to V. Denant. This research was supported by C.N.R.S., France, as part of the GRECO 'Interactions Continent-Ocean' programme.
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Liaaen-Jensen, S., 1977. Algal carotenoids and chemosystematics. In: D.J. Faulkner and W.H. Fenical (Editors), Marine Natural Products Chemistry. Plenum, New York, pp. 239259. Mantoura, R.F.C., 1987. Organic films at the halocline. Nature, 328: 589-590. Mantoura, R.F.C. and Llewellyn, C.A., 1983. The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reverse-phase highperformance liquid chromatography. Anal. Chim. Acta, 151: 297-314. Mantoura, R.F.C. and Woodward, E.M.S., 1983. Conservative behaviour of riverine dissolved organic carbon in the Severn estuary: chemical and geochemical implications. Geochim. Cosmochim. Acta, 47:1293-1310. Morris, A.W., Mantoura, R.F.C., Bale, A.J. and Howland, R.J.M., 1978. Very low salinity regions of estuaries: important sites for chemical and biological reactions. Nature, 274: 678680. Relexans, J.C., Meybeck, M., Billen, G., Brugeaille, M., Etcheber, H. and Somville, M., 1988. Algal and microbial processes involved in particulate organic matter dynamics in the Loire estuary. Estuarine Coastal Shelf Sci., 27: 625-644. Ridout, P.S. and Morris, R.J., 1985. Short-term variations in the pigment composition of a spring phytoplankton bloom from an enclosed experimental ecosystem. Mar. Biol., 87: 7II. Saliot, A., Tronczynski, J., Scribe, P. and Letolle, R., 1988. The application of isotopic and biogeochemical markers to the study of the biochemistry of organic matter in a macrotidal estuary, the Loire, France. Estuarine Coastal Shelf Sci., 27: 645-669. Sekuli6, B., 1989. Estimation of anthropogenic inputs to the Krka River and Estuary (in Croatian). In: National Park Krka, State of Research and Protection of the Ecosystem. Ecological Monograph, Zagreb, 2:153-165. Sholkovitz, E.R., 1976. Flocculation of dissolved organic and inorganic matter during the mixing of river water and sea water. Geochim. Cosmochim. Acta, 40:831-845. gkrivani~, A. and Gr~eti~, Z., 1986. Basic hydrographic parameters and nutrients. In: Longterm Programme for the Pollution Monitoring and Research in the River Krka Estuary and Kornati Archipelago (Adriatic Sea). MED POL - - Phase II. Annual Reports for 1985. Center for Marine Research, Rudjer Bogkovi~ Institute, Zagreb. Svetlici6, V., Zuti6, V. and Tomai~, V., 1991. Estuarine transformation of organic matter: single coalescence events of estuarine surface-active particles. Mar. Chem., 32: 253-267. Tangen, K. and Bj~Srnland, T., 1981. Observations in pigments and morphology of Gyrodinium aureolum Hulburt, a marine dinoflagellate containing 19' hexanoyloxyfucoxanthin as the main carotenoid. J. Plankton Res., 3:389-401. Vernet, M. and Lorenzen, C.J., 1987. The relative abundance of pheophorbide a and pheophytin a in temperate marine waters. Limnol. Oceanogr., 32: 352-358. Vili~i6, D., Legovi~, T. and Zuti~, V., 1989. Vertical distribution of phytoplankton in a stratified estuary. Aquatic Sci., 51: 31-46. Welschemeyer, N.A. and Lorenzen, C.J., 1985. Chlorophyll budgets: zooplankton grazing and phytoplankton growth in a temperate fiord and the Central Pacific Gyres. Limnol. Oceanogr., 30: 1-21. Wollast, R., 1983. Interactions in estuaries and coastal waters. In: B. Bolin and R.B. Cook (Editors), The Major Biogeochemical Cycles and their Interactions, SCOPE 21. Wiley, Chichester, pp. 385-410. Wright, S.W. and Jeffrey, S.W., 1987. Fucoxanthin pigment markers of marine phytoplankton analysed by H.P.L.C. and H.P.T.L.C. Mar. Ecol. Prog. Set., 38: 259-266. Zuti6, V. and Legovi~, T., 1987. A film of organic matter at the freshwater/seawater interface of an estuary. Nature, 328: 612-614.
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Distribution of algal chlorophyll and carotenoid pigments in a stratified estuary: the Krka River, Adriatic Sea V. Denant t, A. Saliot t,* and R.F.C. M a n t o u r a 2 tLaboratoire de Physique et Chimie Marines de l'Universitb Pierre et Marie Curie, UA CNRS 353, Tour 24-25, 4 Place Jussieu, 75252 Paris Cbdex 05 (France) 2plymouth Marine Laboratory, Prospect Place, West Hoe, Plymouth PL 1 3DH (Gt. Britain) (Received September 28, 1989; revision accepted May 10, 1990)
ABSTRACT Denant, V., Saliot, A. and Mantoura, R.F.C., 1991. Distribution of algal chlorophyll and carotenoid pigments in a stratified estuary: the Krka River, Adriatic Sea. Mar. Chem., 32: 285-297. The detailed distribution of algal chlorophyll and carotenoid pigments was determined around the halocline (freshwater-seawater interface) in the Krka Estuary on the east coast of the Adriatic Sea, in May 1988. After collection of water along the estuary, particulate matter was extracted and analyzed for pigments by high-performance liquid chromatography coupled with absorbance and fluorescence detection. Bottom marine waters were characterized by lower chlorophyll a (chl a) concentrations than encountered in surface waters, decreasing downstream from 0.50/tg 1- t to 0.16/zg 1- t at the marine end-member. The highest concentrations of chl a (up to 26.34 #g 1- ~) were found in the interracial layer, an particularly at one station located off the city of ~ibenik, where high inputs of nutrients supported the accumulation of living algae at the halocline. Fucoxanthin was the most abundant carotenoid, which indicates a euryhaline dominance of diatoms in the estuary, whereas the dinoflageUate-derived carotenoid peridinin was confined to the interfacial and bottom saline waters of the inner estuary. High concentrations of alloxanthin and chl b were found in the interfacial layer, which also suggests an accumulation of Cryptophyceae and green algae in the inner estuary. Phaeophorbides showed higher concentrations in bottom waters than in surface waters, whereas the highest concentrations occurred in the interracial layer. These high levels could reflect a density trapping of dead cells in an early degradation state, as suggested by the importance of allomerized chl a and chlorophyUide a vs. total chl a, or of faecal pellets originating from zooplankton grazing in the interfaciai layer.
INTRODUCTION
The importance of acquiring further knowledge of the biogeochemistry of organic matter in estuarine systems, in particular that at the freshwater-seawater interface, withrespect to the high productivity and the various biogeo*Author to whom correspondence should be addressed.
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chemical processes involved in these areas, has been highlighted by several authors (Sholkovitz, 1976; Morris et al., 1978; Cadee, 1982; Wollast, 1983; Mantoura and Woodward, 1983; Ertel et al., 1986; Hedges et al., 1986; Mantoura, 1987; Zuti6 and Legovir, 1987; Lee and Wakeham, 1988, and references therein; Relexans et al., 1988; Saliot et al., 1988; Denant and Saliot, 1990). The freshwater-seawater interface has been described as a filter which is capable of removing living biomass and detritus; this leads to new biological properties associated with bio-accumulation, bio-degradation and, more generally, bio-transformation processes. These processes could result in selective organic film formation, flocculation and sedimentation (Cuker, 1987: Mantoura, 1987; ~uti6 and Legovir, 1987; Svetlici6 et al., 1991 ). In most tidal estuaries, the difficulty encountered in field observations is mainly due to the very variable stratification of waters according to river flow and tidal coefficient. To study in detail the processes that occur in both fresh and marine waters and in the boundary layer, we have chosen the Krka River, which enters the Adriatic Sea. In this stratified karstic estuary, the suspendedmatter load is very low and the freshwater-seawater boundary is highly compressed and stable, and is easily sampled by divers. To study the evolution and the nature of organic matter in both river and marine waters within the estuary, and the effect of the freshwater-seawater interface on natural organic matter derived from phytoplankton, we have focused on the analysis of algal chlorophyll and carotenoid pigments, using high-performance liquid chromatography (HPLC). These organic markers provide chemotaxonomic and physiological information on phytoplankton populations and, more generally, on the extent of degradation of algal-derived organic matter (Jeffrey, 1974; Johansen et al., 1974; Goodwin, 1976; Liaaen-Jensen, 1977; Mantoura and Llewellyn, 1983; Klein and Sournia, 1987; Vernet and Lorenzen, 1987). In this paper, we will attempt to determine the vertical and axial changes in the nature and concentration of pigments that occur (1) in the surface river waters during mixing with seawater in the estuary; (2) in the bottom marine waters, which enter the estuary for a distance of ~ 25 km; and (3) at the freshwater-seawater interface at three representative stations sampled in spring 1988, characterized by a relatively high phytoplanktonic productivity. MATERIAL AND METHODS
Sampling sites The Krka River is a karstic river entering the Adriatic Sea (Fig. 1 ). The Krka River estuary waters are characterized by a low suspension load and low terrigenous inputs; they are poor in nutrients ( ~ v a n i ~ and Gl~etir, 1986 ), with the exception of high phosphate near the harbour area of~ibenik. In this
287
ALGAL CHLOROPHYLL AND CAROTENOID PIGMENTS IN THE KRKA
Skradin Water
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Fig. 1. Krka Estuary, May 1988 cruise: location of sampling sites and distribution of salinity along the estuary. stratified estuary, the boundary layer, which is easily visible and sampled by divers, is always situated in the euphotic zone. Water samples were collected in the Krka Estuary in May 1988 throughout the estuary, from the waterfalls at the head of the estuary to marine waters off Zlarin island. Stations were sampled for surface waters (freshwater at Eo, with an increasing salinity up to the marine point, M.P.; Fig. 1 ), deep marine waters and the boundary layer at stations E2, E3 and E4a (the last station was located off the city of ~ibenik).
Collection of samples Water samples (20 1) were taken by in situ pumping and, for the interface, with the help of a diver. Water was filtered in subdued light a few hours after sampling, on glass fibre filters (Whatman G F / F , 0.7-#m pore size, 47-ram diameter). Filters were precleaned by rinsing with acetone for 24 h in a Soxhlet apparatus. Filters were always kept, even during transport, in glass tubes in a freezer ( T < - 20 ° C), and were analyzed within 1 month after the sampling.
Analysis Each filter, still frozen, was extracted with 3 ml of a mixture of cold acetone and HPLC-grade MilliQ water (9: l, v / v ) in subdued light. Acetone extract (500/A) was sampled with a syringe and added to 150/tl of ion-pairing reagent (solution P) prepared from 1.5 g of tetrabutylammonium acetate and
288 I
V, DENANTETAL. D.V.
11 5
1 4
A
1 Absorbance
5
10 Time
( rain )
11
,D.V
1 13
14
10 luorescence
Time
( mm
)
Fig. 2. Reverse phase H PLC absorbance (2 = 440 nm) and fluorescence ('~excitation = 440 nm: 2emi.ion = 500--700 nm) chromatograms of carotenoids and chlorophyll pigments from an extract of particles collected at station E 3. at 10-m depth. D.V. = dead volume. Peak identities: ( I ) chlorophyll c: (2) peridinin; (3) fucoxanthin-like compound No. 1; (4) fucoxanthin; ( 5 ) fucoxanthin-like compound No. 2; (6) diadinoxanthin; (7) alloxanthin; (8) zeaxanthin/lutein; (9) chlorophyll b; (10) allomerized chlorophyll a; ( 11 ) chlorophyll a; ( 12 ) ~-carotene; (13) phaeophorbide a-like compound; (14) phaeophorbide a.
7.7 g of ammonium acetate made up to 100 ml with water. After thorough mixing, 100/zl were injected into the chromatograph, using a Rlaeodyne model 7125 injector valve. Chromatographic separation was performed following a methodslightly modified from that of Mantoura and Llewellyn ( 1983 ). Separation of pigments was obtained using a 25-cm X 1/4-in. column packed with 5-/1m silica (C18 CDS2, Spherisorb) by linear gradient elution from 100% A to 99% B in 10 min, followed by an isocratic step at 99% B for 12 rain. Solution A was made up of a methanol-water-solutio n P mixture (8" 1 : 1, v / v / v ) . Solution B was made up of methanol-acetone (7: 3, v/v). The HPLC equipment consisted of L.D.C. Milton Roy constametric I and IIt G pumps, a programmable gradient elution system, an L,D.C. Milton Roy Fluoromonitor III fluorescence detector ()~excitation'-- 440 rim; 2em,,io, = 500-700 rim) and
ALGAL CHLOROPHYLL AND CAROTENOID PIGMENTS IN THE KRKA
289
a Beckman 165 UV detector used at 2 = 440 nm. Typical absorbance and fluorescence chromatograms of pigments extracted from particulate matter sampled at station E3 under the boundary at 10-m depth are shown in Fig. 2. The pigments were measured using standards (Mantoura and Llewellyn, 1983 ). Within the conditions used, the following remarks should be made: ( 1 ) two phaeophorbides were characterized, and denoted as phaeophorbide a (peak 14) and phaeophorbide a-like compound (peak 13 ), according to the terminology used by BurkiU et al. ( 1987); (2) two compounds eluting very close to fucoxanthin were separated, and were denoted as fucoxanthin-like compound No. 1 (peak 3) and fucoxanthin-like compound No. 2 (peak 5 ).
Summary of pigment distribution interpretation Chlorophyll a (chl a) is a ubiquitous pigment and can be used as a global algal (freshwater and marine) biomass indicator. Different degradation products from chl a will be considered here. Allomerized chl a, chlorophyllide a, have been found after cell lysis by bacteria (Gieskes et al., 1978 ). Chlorophyllide a is generally considered to be an autolytic degradation product of chl a which is reversibly catalyzed by the enzyme chlorophyllase (Bogorad, 1976); thus, it could indicate the presence of senescent cells (Jeffrey, 1974), although it has been also reported to occur during periods of active growth (Ridout and Morris, 1985 ). The phaeophorbides a are also products of degradation of chl a; phaeophorbide a has been often found in faecal pellets (Jeffrey, 1974). Thus zooplankton grazing is considered to be the major source of this pigment (Welschmeyer and Lorenzen, 1985 ). Fucoxanthin and chl c are indicators of diatoms (Goodwin, 1976 ), whereas peridinin and chl c are synthesized by dinoflagellates (Johansen et al., 1974). Chlorophyll b (chl b) has been commonly ascribed to green algae (Jeffrey, 1974). RESULTS
Characteristics of surface waters Chlorophyll a Figure 3 shows the distribution of chl a concentrations along the estuary. Chl a concentrations of surface waters increase from < 1 #g 1- ~ at Eo (pure river water) and E2 (S< 30/00), at the head of the estuary, and reach a maxim u m of 2.05/zg 1-~ at station E4a (S=7%o) near ~ibenik. Thereafter, chl a decreases to 1.08/zg 1- ~at station C2 ( S = 25%o) and 0.38/tg l - ~at the marine end-member (S~ 370/00).
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Fig. 3. Krka Estuary, May 1988: distribution ofchl a concentrations expressed in #g 1- l in surface water, boundary layer and bottom water. Chl a concentration at station E4, boundary layer is out of scale (26.34/tg l - ] ). 0.6-
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Fig. 4. Krka Estuary, May 1988: distributions of allomerized chl a, chlorophyllide a, phaeophorbide a and phaeophorbide a-like compound in surface water, boundary layer and bottom water.
Degradation productsfrom chlorophyllpigments Distributions o f allomerized chl a and chlorophyllide a are very similar to that o f chl a (Fig. 4). Their concentrations are low in the variation ranges 0.01-0.06/tg l - ' and 0.00-0.08/tg 1-1, respectively. These two degradation products represent < 5% o f total chl a.
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ALGAL CHLOROPHYLL AND CAROTENOID PIGMENTS IN THE KRKA
Neither phaeophorbide a nor phaeophorbide a-like compound has been detected in the low-salinity section of the estuary ( S < 3%0). With the increasing influence of marine waters, these two compounds appear, and they reach their highest concentration at the marine point, where they represent up to 16 and 34% of chl a, respectively (Fig. 4).
Taxonomic pigments Fucoxanthin is the predominant carotenoid identified for all samples. Its concentration varies from 0.07 to 0.32/zg 1-1; the minimum value corresponds to the marine reference (Fig. 5 ). Peridinin was not detected in any of the surface samples. The distributions of alloxanthin and chl b are close to that ofchl a. The highest concentrations occur at station E4a - - 0.27 and 0.14 /zg l- 1, respectively.
Characteristics of bottom (marine) waters Chlorophyll a Water samples below the halocline/thermocline are characterized by lower chl a concentrations than in surface waters; the concentration decreases
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Fig. 5. Krka Estuary, May 1988: distributions of concentrations of taxonomic pigments, fucoxanthin, peridinin, alloxanthin and chlorophyll b in surface water, boundary layer and bottom water. (Note the change of scale used for pigment concentration ( 0 - 6 pg l - 1) for the E4a interface sample. )
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downstream, from 0.50/~g 1-J at station Eo to 0.16 ~tg 1-J at the marine reference (Fig. 3).
Degradation products from chlorophyllpigments Allomerized chl a has very low concentrations which vary in a narrow range (0.00-0.01/~g 1-J ), whereas chlorophyllide a was not detectable (Fig. 4 ). Phaeophorbides a distributions are most interesting. Phaeophorbide a is m a x i m u m at station E3 (0.27/tg 1- J ) but has a much lower concentration variation (in the range 0.00-0.01/~g 1-j ) for other samples, Phaeophorbide a-like c o m p o u n d has two concentration maxima, which occur at stations E 3 and C2.
Taxonomic pigments Fucoxanthin is the predominant carotenoid, but at a lower level than observed in surface waters (Fig. 5 ). Peridinin is present only at stations E2 and E3. Alloxanthin and chl b are present at low concentrations, which decrease downstream. Two pigments that have a structure close to fucoxanthin which are absent in surface waters are detected here: fucoxanthin-like c o m p o u n d No. 1, present only in samples where peridinin occurs, and fucoxanthin-like c o m p o u n d No. 2 at stations E2, E3, C2 and the marine reference point, where phaeophorbide a-like c o m p o u n d was also detected.
Characteristics of the freshwater-marine water interface Chlorophyll a Chl a concentrations are higher than those observed in surface waters (Fig. 3 ). A very high value of 26.34/tg 1-J was observed at station E4a.
Degradation products from chlorophyllpigments Allomerized chl a and chlorophyllide a show a remarkable increase in terms of absolute concentration (Fig. 4), but not in terms of percentage of total chl a, which has a relative importance of < 2%. Phaeophorbide a-like compound is significantly present at stations E2 and E3, where it accounts for ~ 27% ofchl a.
Taxonomic pigments As for other samples from both surface and bottom layers, fucoxanthin predominates (Fig. 5 ), but here alloxanthin and chlorophyll b are present at relatively high levels, e.g. 1.39 and 0.79/~g 1- J at station E4a. Peridinin and fucoxanthin-like c o m p o u n d No, 1 are present only at station E2, whereas fucoxanthin-like c o m p o u n d No. 2 has a m a x i m u m a t s t a t i o n E3: 0.17 /tg1-1.
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DISCUSSION Considering the three complete vertical profiles obtained at stations E2, E3 and E4a, the freshwater-seawater interface is characterized first by a marked enrichment in chl a. This enrichment has been previously observed during winter conditions in November 1984 (Vili~i~ et al., 1989 ) and in March 1987 (Denant, 1988), and during spring conditions in April 1985 (Vili~i~ et al., 1989 ). Vili~i~ et al. (1989) have documented the vertical distribution ofphytoplankton, and have shown that the selective accumulation of larger phytoplankton cells and the associated high chl a concentrations in the interfacial layer are a consequence of the accumulation of viable freshwater phytoplankton cells in the upper boundary of the interface. Chl a concentrations are highest in the film, and particularly at station E4a, for both winter and spring conditions. These high levels can be easily explained by the proximity to the city of ~ibenik (Fig. l ), as large nutrient inputs, especially of phosphate ( 106 mol year-1; Sekuli~, 1989), are introduced into the estuary from sewage effluents and harbor activities. Distribution profiles of allomerized chl a and chlorophyUide a are close to that of chl a. Their concentrations relative to the parent compound chl a are small, which suggests that, at this time of year, the degradation of organic material of phytoplanktonic origin through senescence of cells and lysis by bacteria is not pronounced. On the other hand, the opposite was observed during winter conditions, when high concentrations of chlorophyllide a occurred at the interface, representing 10-12% of total chl a. However, the possibility of an artefactual formation of chlorophyllide a during the filtration of water samples could not be ruled out (Jeffrey and Hallegraeff, 1987), although the same precautions have been taken during the two cruises. Phaeophytin a was not detected. Two hypotheses can be proposed to explain this: there may be no formation of phaeophytin a in natural conditions, or there may be a rapid transformation into phaeophorbide a. Two phaeophorbides a were detected. Phaeophorbide a-like compound is always predominant over phaeophorbide a, and has a higher concentration in bottom marine waters than in surface waters, whereas the highest concentrations occur in the interfacial layer. Vilici~ et al. ( 1989 ) have observed such an accumulation of dead phytoplankton cells in the interfacial layer. These vertical profiles could be explained by settling followed by density trapping, in the organic film that develops at the interfacial layer, of particles enriched in phytoplankton material, such as faecal pellets that originate from the grazing of phytoplankton by freshwater zooplanktonic organisms. Such particles are known to be enriched in compounds derived from the degradation of chlorophyll and other recently biosynthesized organic material. Phaeophorbide alike compound has been interpreted as an indicator of a more advanced degradation state of chl a (Vernet and Lorenzen, 1987). The value of the ratio
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Phaeo a-like c o m p o u n d / P h a e o a is in the range 1.5-2.2 for surface marine waters. Bottom waters, which are older, as they are transported from the sea into the estuary on a time-scale of the order of a few years (Martin et al., personal communication, 1989 ), are characterized by values of the ratio Phaeo a-like c o m p o u n d / P h a e o a that increase with the residence time of waters: 0.7 at station E 3 and 5 at station E2. Some interesting features appear when we consider the distribution of chemotaxonomically specific pigments. Fucoxanthin is the principal carotenoid pigment, reflecting the wide distribution of diatoms in these waters (D. Vilici6, personal communication, 1989). The abundance of diatoms varies in the following range: 2.67 × 105 cells 1- J at station Eo; between 2.53 × 105 (E2) and 9.4 × 104 (C:) cells 1- ~ for surface waters; and between 5 × 103 (E3) and 5.1 × 104 (C2) cells 1- ~for bottom waters. There is no systematic relationship between the concentration of fucoxanthin and the abundance of diatoms, whereas such a relation was observed during winter conditions. Peridin in was detected only at station E2 and E 3. This restricted distribution of dinoflagellate in estuarine environments was described by Incze and Yentsch in 1981. It was also observed during winter conditions in March 1987 (Denant, 1988 ). Dinoflagellates here develop in interfacial and bottom waters, where the difference of salinity between surface and bottom waters always exceeds 35%0, at the end of the saline front. C o m p o u n d s derived from or having a structure close to fucoxanthin have been previously observed on HPLC and include hexanoyl- and butanoyl-oxyfucoxanthin esters. They are more uniquely synthesized by various species of prymnesiophyte phytoplankton (Gieskes and Kraay, 1983. 1986a, b; Wright and Jeffrey, 1987; Gieskes et al., 1988 ). Two compounds that have a fucoxanthin-type structure have been detected. Fucoxanthin-like c o m p o u n d No. 1 shows an interesting distribution, as it is present only in the samples where peridinin is observed. It is commonly assumed that dinoflagellates do not synthesize simultaneously peridinin and fucoxanthin-derived compounds (Tangen and Bjtirnland, 1981; Wright and Jeffrey, 1987 ). It is probable that the fucoxanthin-like c o m p o u n d No. 1 originates from species that are not dinoflagetlates but that live in close association with them. The fucoxanthin-like c o m p o u n d No. 2 is always found in association with phaeophorbide a-like c o m p o u n d and thus could derive from the degradation of fucoxanthin. High concentrations of alloxanthin and chlorophyll b found in the interracial layer suggest an accumulation of Cryptophyceae and green algae relative to river and marine waters. These two algal classes appear to develop in the estuary between station Eo and Cz, but not in marine waters offthe estuary.
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CONCLUSION
The stratified Krka Estuary could be considered as a unique model for studying the respective impacts of riverine and marine processes on the evolution of phytogenic organic matter. In May 1988, pigment concentrations varied over a large range along the estuary. Chl a concentrations of surface waters largely depend on the mixing between fresh and saline waters. The same tendency is observed for allomerized chl a and chlorophyUide a. Chl a concentrations of marine waters entering the estuary increase from the marine environment to the inner estuary, which suggests a marked influence of surface waters on the productivity of bottom waters, and possible exchange of nutrients across the interface. Other pigment concentrations, such as for fucoxanthin, peridinin, alloxanthin and chl b, reflect the specific distribution of predominant phytoplankton species such as diatoms, dinoflagellates, Cryptophyceae and green algae, respectively. Compounds with a fucoxanthin-type structure show interesting associations: one compound, which correlates with peridinin, suggests the potential presence of phytoplankton species living in close association with dinoflagellates; the other correlates with phaeophorbide a-like compound and thus originates from the degradation of diatom-derived fucoxanthin. The interfacial layer thus appears to be a biogeochemically very active zone for phytogenic organic matter. The highest chl a concentrations occur in the interfacial layer, reflecting the accumulation of living phytoplankton. Accumulation at the boundary is observed for different pigments related to different phytoplankton species, and occurs at different sites of the estuary, depending on the salinity gradient between surface and bottom marine waters: fucoxanthin occurs in high concentration throughout the estuary, as diatoms predominate in both riverine and saline waters. Alloxanthin and chl b are accumulated in the inner estuary, whereas peridinin is found only in a very few bottom and interface samples, reflecting a more patchy distribution of dinoflagellates. The interface appears also to be an efficient trap for dead cells in faecal pellets of zooplankton, as suggested by the relative accumulation of allomerized chl a, chlorophyllide a and phaeophorbides. ACKNOWLEDGEMENTS
We thank Dr. Vera 7.utir, Dr. G. Cauwet and Dr. J.M. Martin, coordinators of the French-Yugoslavian programme on the Krka River, and C. Llewellyn (Plymouth Marine Laboratory ) for training help to V. Denant. This research was supported by C.N.R.S., France, as part of the GRECO 'Interactions Continent-Ocean' programme.
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