- Email: [email protected]
Plant Pigments as Biomarkers of Organic Matter Sources in Sediments and Coastal Waters of Cyprus (eastern Mediterranean)
Estuarine, Coastal and Shelf Science (1996) 42, 103–115
Plant Pigments as Biomarkers of Organic Matter Sources in Sediments and Coastal Waters of Cyprus (eastern Mediterranean)
Thomas S. Bianchia, Andreas Demetropoulosb, Myroula Hadjichristophoroub, Marina Argyroub, M. Baskaranc, and Corey Lamberta a
Department of Ecology, Evolution and Organismal Biology, Tulane University, New Orleans, Louisiana 70118 U.S.A., bMinistry of Agriculture and Natural Resources, Department of Fisheries, 13 Aeolou Street, Nicosia, Cyprus, and c Department of Oceanography, Texas A&M University, Galveston, Texas 77553, U.S.A. Received 2 August 1994 and in revised form 10 October 1994
Keywords: plant pigments; radionuclides; sedimentation; Cyprus Plant pigments (chlorophylls and cartenoids) and radionuclides (226Ra and 210 Pb) were measured in the coastal waters and sediments off the coast of Cyprus (eastern Mediterranean), for the first time, in June and July 1993. Some of the lowest concentrations of chlorophyll a (10–90 ng l "1) were found in these highly oliogotrophic waters. Based on the presence of chlorophyll b and zeaxanthin, it appeared that chlorophytes, cyanobacteria, and prochlorophytes were the dominant phytoplankton classes. The phytoplankton assemblage off the coast of Cyprus was not very different when compared to the deeper regions of the eastern Mediterranean. However, there were significantly higher concentrations of pigments in sediments principally due to seagrasses and macroalgae. In fact the Red Sea migrant Caulerpa racemosa appears to be expanding its areal coverage along the coastline; it remains to be tested whether this is related to enhanced nutrient inputs or to inherent differences in the life-history characteristics of this migrant vs. native species. The very low sedimentation rates observed at all stations (0·38–0·78 mm year "1) further suggested that input from the water column to the sediments was minimal. The land-margin effects of Cyprus on the surrounding oligotrophic waters of the eastern Mediterranean appeared to only affect the composition and distribution of benthic macrophytes, with very little change in the phytoplankton assemblages. ? 1996 Academic Press Limited
Introduction In past years, much of the research using plant pigments as biomarkers of organic matter sources in the sediments and water column of the Mediterranean has centered on western regions (Plante et al., 1986; Klein & Sournia, 1987; Vaulot et al., 1990). Only recently have some measurements been made on phytoplankton biomass in the eastern Mediterranean; however, these studies have not used high performance liquid 0272–7714/96/010103+13 $12.00/0
? 1996 Academic Press Limited
104
T. S. Bianchi et al.
chromatography (HPLC) techniques and thus, have been restricted to measuring only chlorophyll a (Azov, 1986; Krom et al., 1993). While the western Mediterranean is also considered to be oligotrophic with low chlorophyll a values in summer ranging from 0·05 to 0·5 ìg l "1, many of the northern coastal margins experience massive phytoplankton blooms because of inputs from nutrient-rich rivers (Jacques et al., 1973; Klein & Sournia, 1987). Recent measurements in the south-eastern Mediterranean reveal that prevailing oligotrophic conditions result in low chlorophyll a concentrations ranging from 0·1 to 0·2 ìg l "1 (Krom et al., 1991). Changes in phytoplankton biomass and composition along land-margin ecosystems in this highly oligotrophic basin are likely to be very different than in the western Mediterranean. Cyprus is the easternmost Mediterranean island located in the Levantine basin of the eastern Mediterranean. The eastern Mediterranean is located east of the Sicilian Straights and comprises the Ionian and Levantine basins and the Adriatic and Aegean Seas. Compared with other regions of the Mediterranean, this is the most poorly studied, particularly the Levantine Basin. To date, much of the literature that exists on the eastern Mediterranean has centred on its physical oceanography [i.e. Physical Oceanography of the Eastern Mediterranean (POEM) project] (Hecht et al., 1988; Malanotte-Rizzoli & Hecht 1988; Millot 1992; Malanotte-Rizzoli & Bergamasco 1992; Robinson & Golnaraghi 1992). In fact, the Levantine intermediate water (LIW) formed in the basin, consists of dense salty water that is highly oligotrophic (Hecht et al., 1988). Other studies carried out in the deep waters of the Levantine basin have shown that primary productivity is limited by phosphorus (P) (Krom et al., 1991, 1992). Similarly, coastal waters off the coast of Israel also appear to be P-limited (Azov, 1986). Cyprus, the only large island situated in the eastern Levant, serves as an excellent site for examining land-margin interactions because of its isolation from significant riverine inputs. In this study, we used plant pigments as biomarkers to determine, for the first time, the dominant classes of phytoplankton in the coastal waters of Cyprus. We also used pigments to determine the dominant sources of carbon input to surficial sediments. Excess 210Pb concentrations in the sediments were also used to determine sedimentation rates. Methods and materials Study area and sampling Cyprus has an area of 9251 km2, the nearest distance to a coast is 75 km to southern Turkey, 102 km to Syria, and 384 km to Egypt. Water samples and sediments were collected for pigments aboard the RV Alkion at four regional stations along the coast of Cyprus in June and July 1993 (Figure 1). A shallow (25 m) and deep (50 m) station were sampled at each of the regional stations twice a month. Water samples for salinity and temperature profiles were taken at the deep stations using Nansen bottles. Surficial sediments were collected using an ‘ Orange Peel ’ grab sampler; sediment cores, for radionuclide analysis, were taken using a Piston-corer. Percent organic matter was measured by weight loss upon combustion at 500 )C for 24 h. Plant pigments Aliquots of 200–300 ml taken from the water sample were filtered through glass fiber filters (Whatman GF/F filters, 25 mm diameter, nominal pore size of 0·7 ìm) for plant pigment analysis (see Bianchi et al., 1993 for further details on extraction procedures).
Plant pigments as biomarkers
105
33°00'
Turkey
Greece Iráklion Rhodes Crete
Cyprus
Mediterranean Sea
35°30'
Syria Lebanon Beirut
km 100 0 100 200 300 400 Haifa Tel Aviv 100 200 Jordan Port Said Israel Alexandria
35°30'
miles100 50 0 Libya
35°00'
34°00'
Cyprus Famagusta
Latsi
35°00'
1 0 1 101520 km
Pafos Limassol
33°00'
5
0
10 15 20 miles
34°00'
Figure 1. Map of Cyprus showing the four regional stations sampled in June and July 1993. Although not shown in map there were two stations (25 m and 50 m depth) sampled at each regional station.
Due to time restrictions, we were not able to compare GF/F filters with 0·2 ìm pore size filters. Canthaxanthin was placed in all acetone extracts as an internal standard. Reverse-phase analysis was conducted using the method of Wright et al. (1991). A Waters Model 610 solvent delivery system was coupled with dual-channel detection using a Waters Model 996 photodiode array detector set at 438 nm for absorbance and a Milton–Roy fluorescence detector with excitation set at 440 nm and emission at >600 nm. The injector was connected via a guard column to a reverse-phase C18 Alltech adsorbosphere column (5 ìm particle size; 250 mm#4·6 mm i.d.). After injection (100 ìl) a gradient program (1 ml min "1) begins isocratically with mobile phase A (80:20 methanol: 0·5 M ammonium acetate, aq.; pH 7·2 v/v) which then ramps to 100% mobile phase B in 4 min (90:10 acetonitrile:HPLC grade water v/v) and then changes to 20% B and 80% mobile phase C (100% ethyl acetate) in 14 min. This is followed by a return to 100% B in 3 min with final ramping to 100% A in 3 min. This gradient was slightly modified for sediments resulting in a longer run time of 45 min. The method allowed for adequate resolution of dominant peaks of interest (Figure 2). High purity HPLC standards for chlorophylls a and b and á, â-carotenes were obtained from Sigma Co. Standards of the following carotenoids were kindly provided by Hoffman LaRoche Co., Basel, Switzerland: fucoxanthin; lutein; zeaxanthin; peridinin. Radionuclides Dried sediment core sections were pulverised using an agate mortar and pestle. Two grams of the powered samples were then transferred into microwave digestion vessels, to
106
T. S. Bianchi et al.
Relative absorbance
d
c
a
0
b
15.00 Retention time (min)
30.00
Figure 2. Absorbance chromatogram from a water sample collected at Famagusta (50 m) in June 1993. The identified pigment peaks are as follows: (a) Fucoxanthin; (b) Zeaxanthin; (c) Chlorophyll b; (d) Chlorophyll a.
which 10 ml of concentrated HF and 15 ml of concentrated HNO3 acids (trace metal grade) were added. A known amount of 209Po spike was added to the powder. Sediment digestions were accomplished using a commercial microwave digestion system (model CEMR -MD-81D). The contents in the digestion vessels were heated in the microwave at a maximum pressure of 90 p.s.i. for about 3 h. The solutions were then transferred to acid-cleaned teflon beakers containing 1 M HCL and taken to dryness on a hot plate. Polonium was electroplated onto silver planchets following the method of Flynn (1968), and then assayed for 210Pb by using an alpha spectrometer with a surface detector coupled to a Canberra S-100 multichannel analyser. The sediment samples were packed into 10-ml gamma counting vials and counted on a high purity Ge well-detector coupled to a Canberra S-100 multichannel analyser. The peak analysis of 226Ra (352 keV) was done using SPECTRAN-AT peak analysis software. The gamma counting equipment was calibrated with fly ash standards obtained from the Environmental Measurement Laboratory. Sedimentation rates were calculated using the methods described in Krishnaswami et al. (1971).
Plant pigments as biomarkers
107
Statistical analyses An Fmax was used prior to ANOVA analysis to check for homogeneity of variances. A two-way ANOVA was used to test for significant effects of station and depth on pigments. When ANOVA differences were significant, a Scheffe multiple range test was performed to detect for differences between means. Correlation analyses were performed using Pearsons product moment correlation matrix. Results Physicochemical data During June and July 1993, surface water temperatures (among all stations) ranged between 20–28 )C as compared to bottom temperatures which ranged between 16–20 )C. The salinity profiles were not highly stratified with the average salinity being maintained at c. 39. Transparency secchi disk readings ranged between 21–39 m with the highest clarity at the Latsi station. Water column pigments In June 1993, chlorophyll a concentrations ranged between 16–90 ng l "1 at all four regional stations, with a similar range of 10–90 ng l "1 in July 1993 (Figures 3–6). The highest concentrations occurred at the Pafos and Limassol stations, with the lowest measurable concentrations at Famagusta and Latsi. In June 1993, the subsurface chlorophyll a maxima generally occurred between 20–30 m at the 50-m stations. In July 1993, chlorophyll a concentrations were generally uniform throughout the water column with minor increases at depth at Famagusta and Latsi. Overall, the chlorophyll a concentrations in July, between 0–10 m, were significantly (P<0·05) lower than in June, except for Latsi. Chlorophyll b concentrations ranged between 0–76 ng l "1 in June 1993 and 2–55 ng l "1 in July 1993. Concentrations of the accessory pigment chlorophyll b, which is abundant in chlorophytes, were significantly correlated (P<0·05) with chlorophyll a concentrations at all stations in June and July. The highest concentrations (P<0·05) of chlorophyll b occurred at the Limassol station. Concentrations of fucoxanthin, a carotenoid commonly found in diatoms, ranged between 0–10 ng l "1 in June 1993 and only 0–3 ng l "1 in July 1993; the highest concentration (P<0·05) was found at the 50 m Pafos station. Zeaxanthin, a carotenoid found in cyanobacteria, had a concentration range of 0–14 ng l "1 in June 1993 and 0–16 ng l "1 in July 1993. Peridinin, a carotenoid marker for dinoflagellates, was generally below limits of detection in June 1993, however, it did reach detectable concentrations at stations Limassol and Pafos in July 1993 that ranged between 3–18 ng l "1 (data not shown). Concentrations of á-carotene were significantly higher (P<0·05) (3–31 ng l "1) than â-carotene (0–5 ng l "1) at all stations (data not shown). Sedimentary pigments Chlorophyll a concentrations in surface sediments (at all four stations) ranged between 0·05–0·46 ìg g dry sediment "1 and 0·03–0·37 ìg g dry sediment "1 in June and July 1993, respectively (Table 1). In July 1993, concentrations of chlorophyll a were always significantly (P<0·05) higher at the 25-m stations than at the 50-m stations, except for Famagusta. Concentrations of chlorophyll a were highest (P<0·05) at the Latsi and Famagusta stations in July 1993. Chlorophyll b concentrations ranged between 0·008–0·26 ìg g dry sediment "1 and 0·01–0·22 ìg g dry sediment "1 in June and July
108
T. S. Bianchi et al.
–1
–1
Pigment concentration (ng l ) 0
20
40
60
Pigment concentration (ng l ) 0
80 100 120
20
40
60
80 100 120
June 1993, 50 m 10
Depth (m)
Depth (m)
5
10
15
June 1993, 25 m
20
20
30
40
25
50
Pigment concentration (ng l–1) 0
20
40
60
Pigment concentration (ng l–1) 0
80 100 120
July 1993, 25 m
60
80 100 120
10
Depth (m)
Depth (m)
15
40
July 1993, 50 m
5
10
20
20
30
20
40
25
50
Figure 3. Water column pigment profiles (ng l "1) at the Famagusta station (25 m and 50 m) off the coast of Cyprus in June and July 1993. -, Chlorophyll a; ., Chlorophyll b; 4, Fucoxanthin; :, Zeaxanthin.
1993, respectively. Fucoxanthin and zeaxanthin concentrations were generally low with concentrations ranging between 0·01–0·26 ìg g dry sediment "1 and 0·01– 0·16 ìg g dry sediment "1 in June and July 1993, respectively. Lutein, a carotenoid commonly found in chlorophytes, ranged between 0·02–0·32 ìg g dry sediment "1 with highest concentration occurring at the Limassol station (data not shown). Sedimentation rates There appeared to be no sediment mixing at all four stations, the excess 210Pb penetration depths varied between 5–8 cm. The linear sedimentation rates at the four
Plant pigments as biomarkers
109
–1
–1
Pigment concentration (ng l ) 0
20
40
60
Pigment concentration (ng l ) 0
80 100 120
20
June 1993, 25 m
Depth (m)
Depth (m)
80 100 120
10
10
15
20
30
20
40
25
50
Pigment concentration (ng l–1) 20
40
60
Pigment concentration (ng l–1) 0
80 100 120
July 1993, 25 m
15
40
60
80 100 120
10
Depth (m)
10
20
July 1993, 50 m
5
Depth (m)
60
June 1993, 50 m
5
0
40
20
30
20
40
25
50
Figure 4. Water column pigment profiles (ng l "1) at the Limassol station (25 m and 50 m) off the coast of Cyprus in June and July 1993. -, Chlorophyll a; ., Chlorophyll b; 4, Fucoxanthin; :, Zeaxanthin.
stations varied between 0·38–0·70 mm year "1. The parent-supported 226Ra concentration varied between 0·40–0·76 dpm g "1. The linear sedimentation rates at Latsi and Pafos were comparable, 0 38 mm year "1 and 0·44 mm year "1, respectively. The fastest sedimentation rate was observed at Limassol, 0·70 mm year "1, the sedimentation rate at Famagusta was 0·58 mm year "1. Discussion Organic matter sources in the water column Chlorophyll a concentrations (commonly used to estimate primary production biomass) in the surface waters off the coast of Cyprus are among the lowest values (10–90 ng l "1)
110
T. S. Bianchi et al.
–1
–1
Pigment concentration (ng l ) 0
20
40
60
Pigment concentration (ng l ) 0
80 100 120
40
60
80 100 120
10
10
15
June 1993, 25 m
Depth (m)
Depth (m)
5
20
20
20
30
40 June 1993, 50 m
25
50
Pigment concentration (ng l–1) 0
20
40
60
Pigment concentration (ng l–1) 0
80 100 120
July 1993, 25 m
60
80 100 120
10
Depth (m)
Depth (m)
15
40
July 1993, 50 m
5
10
20
20
30
20
40
25
50
Figure 5. Water column pigment profiles (ng l "1) at the Pafos station (25 m and 50 m) off the coast of Cyprus in June and July 1993. -, Chlorophyll a; ., Chlorophyll b; 4, Fucoxanthin; :, Zeaxanthin.
ever observed in nearshore waters. The highest concentrations of chlorophyll a, which occurred at the Pafos and Limassol stations, are still remarkably lower than the range of concentrations (0·15–0·25 ìg l "1) observed in deeper oligotrophic waters of the eastern Mediterranean (Krom et al., 1991). The highest chlorophyll a inventories also occurred at Limassol and Pafos (50-m stations) in June 1993; the lowest occurred at Pafos (25-m station) in July 1993 (Table 2). The highest chlorophyll a inventory at Limassol (2·17 mg m "2) may be related to a greater input of anthropogenic nutrients (i.e. agriculture, sewage and industry) at this station, which is the largest coastal city in Cyprus. In a recent study of the coastal aquifer, between Limassol and Famagusta, it was concluded that fertilizers used in excess by farmers (primarily for potatoes and citrus)
Plant pigments as biomarkers
111
–1
–1
Pigment concentration (ng l ) 0
20
40
60
Pigment concentration (ng l ) 0
80 100 120
20
10
15
20
30
20
40
25
50
Pigment concentration (ng l–1) 20
40
60
Pigment concentration (ng l–1) 0
80 100 120
July 1993, 25 m
40
60
80 100 120
10
Depth (m)
Depth (m)
20
July 1993, 50 m
5
15
80 100 120
10
Depth (m)
Depth (m)
5
10
60
June 1993, 50 m
June 1993, 25 m
0
40
20
30
20
40
25
50
Figure 6. Water column pigment profiles (ng l "1) at the Latsi station (25 m and 50 m) off the coast of Cyprus in June and July 1993. -, Chlorophyll a; ., Chlorophyll b; 4, Fucoxanthin; :, Zeaxanthin.
are clearly contributing to the developing eutrophication problems along the coast (Christodoulides, 1994). For example, based on a total groundwater outflow of 1000 m3 day "1 for this region, the total annual nutrient outflows of nitrate and phosphorous to the sea are 20 metric tons and 50 kg, respectively (Christodoulides, 1994). The significantly lower chlorophyll a concentrations observed in July (between 0–10 m) are probably due to the result of changes in nutrients, rather than grazing effects because of the very low phaeopigment concentrations observed at all stations: chlorophyllides ranged between 0·05–0·78 ng l "1, total phaeophytins ranged between 0·12–1·12 ng l "1, and total phaeophorbides ranged between 1·14–5·13 ng l "1.
112
T. S. Bianchi et al.
T 1. Percent organic matter and plant pigment concentrations (ìg g "1 dry sediment "1) in surficial sediments (0–2 cm) collected from four locations at 25 m and 50 m depths, in July 1993 (only 50 m depth for June 1993) off the coast of Cyprus in the eastern Mediterranean, SE= &.
Date June 1993 June 1993 June 1993 June 1993 July 1993 July 1993 July 1993 July 1993 July 1993 July 1993 July 1993 July 1993
Station/depth
% Organic matter
Famagusta (50 m) Limassol (50–m) Pafos (50 m) Latsi (50 m) Famagusta (25 m) Famagusta (50 m) Limassol (25 m) Limassol (50 m) Pafos (25 m) Pafos (50 m) Latsi (25 m) Latsi (50 m)
9·7&1·9 9·6&1·0 10·1&2·1 13·2&3·1 9·7&1·4 10·3&1·1 6·8&2·2 9·3&2·1 6·8&2·6 7·4&1·9 6·2&1·3 9·7&2·7
Chlorophyll a
Chlorophyll b (ìg g
0·46&0·12 0·05&0·01 0 12&0·03 0·09&0·01 0·28&0·01 0·34&0·05 0·23&0·02 0·12&0·002 0·19&0·003 0·05&0·004 0·37&0·03 0·03&0·003
"1
Fucoxanthin
dry sediment
0·09&0·01 0·26&0·001 0·05&0·004 0·008&0·002 0·04&0·003 0·07&0·03 0·11&0·001 0·02&0·001 0·22&0·001 0·01&0·001 0·07&0·01 0·01&0·001
Zeaxanthin
"1
)
0·03&0·001 0·01&0·001 0·26&0·01 0·03&0·01 0·11&0·002 0·18&0·03 0·14&0·004 0·08&0·01 0·08&0·01 0·03&0·01 0·21&0·004 0·13&0·002
0·05&0·01 0·16&0·007 0·01&0·002 0·01&0 002 0·03&0·001 0·07&0·02 0·07&0·02 0·03&0·01 0·01&0·001 0·02&0·001 0·03&0·003 0·01&0·001
T 2. Chlorophyll a inventories at four stations in the coastal waters of Cyprus in June and July 1993
Date
Station/depth (m)
Chlorophyll a inventory (mg m "2)
June 1993 June 1993 June 1993 June 1993 June 1993 June 1993 June 1993 June 1993 July 1993 July 1993 July 1993 July 1993 July 1993 July 1993 July 1993 July 1993
Famagusta (25 m) Famagusta (50 m) Limassol (25 m) Limassol (50 m) Pafos (25 m) Pafos (50 m) Latsi (25 m) Latsi (50 m) Famagusta (25 m) Famagusta (50 m) Limassol (25 m) Limassol (50 m) Pafos (25 m) Pafos (50 m) Latsi (25 m) Latsi (50 m)
1·21 1·02 1·48 2·17 1·29 2·05 0·89 1·76 0·66 1·21 0·63 0·78 0·38 0·93 0·44 1·00
Based on relatively higher concentrations of the accessory pigments zeaxanthin and chlorophyll b, it appears that most of the phytoplankton in the coastal waters of Cyprus is comprised of chlorophytes, cyanobacteria, and prochlorophytes. These classes of phytoplankton typically dominate in oligotrophic oceanic waters (Chisholm et al., 1988; Ondrusek et al., 1991). While chlorophyll b is a marker for chlorophytes and zeaxanthin is commonly found in cyanobacteria, prochlorophytes have been found to contain both of these pigments (Bidigare et al., 1986; Chisholm et al., 1988; Ondrusek et al., 1991). Thus, the presence of prochlorophytes makes it very difficult to separate the relative importance of chlorophytes vs. cyanobacteria. Prochlorophytes are prokaryotic picoplankton commonly found in oligotrophic oceanic waters; they are characterized as
Plant pigments as biomarkers
113
having chlorophyll b, divinyl chlorophyll a (substituted for chlorophyll a), zeaxanthin, and á-carotene (substituted for â-carotene) (Chisholm et al., 1988). Recent work has shown that prochlorophytes are not restricted to oceanic regimes and have been found to be important in the north-western Mediterranean as well as the Gulf of Mexico (Vaulot et al., 1990; Vaulot & Partensky, 1992; Bianchi et al., 1994). The typical chlorophyll a/zeaxanthin ratio of prochlorophytes is c. 3·0; the range of ratios found at the Cyprus stations (at the 50-m station in the upper 10 m) was between 1·05–15·93 (mean&6·18 SD&4·1). Except for a few high ratios at Latsi and Pafos, most of the ratios fall within the range of 2–6, very close to that of cyanobacteria and prochlorophytes. The chlorophyll a/zeaxanthin ratios, at the 25-m station and in the lower water column at both depths, tend to be higher presumably due to detrital macrophytic inputs from resuspended bottom materials. The ratios of older macrophytic detritus are higher due to differences in the decay kinetics of chlorophyll a and zeaxanthin, since chlorophyll a decays more rapidly (Bianchi & Findlay, 1991). The reverse-phase HPLC method used here does not resolve divinyl chlorophyll a, thus, we were not able to determine the relative importance of prochlorophytes. However, the dominance of á-carotene vs. â-carotene in these waters further suggests that prochlorophytes are likely to be a component of the phytoplankton. The presence of fucoxanthin in very low concentrations suggests that diatoms contributed only marginally to the total phytoplankton assemblage in June and July 1993. If prochlorophytes are a significant component of the phytoplankton assemblage, using GF/F filters would likely result in an underestimation of chlorophyll a concentrations because of loss through the pores; recent studies show that 0·2 ìm pore size filters can result in significant differences (Dickson & Wheeler, 1993; Bianchi et al., 1994). Organic matter sources in sediments Concentrations of chlorophyll a in sediments along the coast of Cyprus were always found to be substantially higher than those found in the water column—due to high densities of macroalgae and seagrasses. The very low sedimentation rates observed at all stations further suggests that there is low input from the water column to the sediments. The range of sedimentation rates observed here (0·38–0·70 mm year "1) are considerably lower than that found in most coastal regions (Appleby & Oldfield, 1992). The high incidence of light to these sediments (estimated by secchi disk), at both the 25-m and 50-m stations, should allow for rapid expansion phytobenthos—if pulsed with nutrients. The highest chlorophyll a concentrations in sediments occurred at Famagusta and Latsi, however, the density of macrophytes in these regions may not be significantly greater than the other stations. This inconsistency can likely be explained by high spatial heterogeneity commonly found within these benthic communities (Hadjichristoforou & Argyrou, 1993a). The availability of nutrients to these macrophytes will also be driven in part by the regeneration of nutrients from decaying macrophyte detritus. However, it is likely that anthropogenic inputs from agriculture and tourism may be more of a controlling factor (Christodoulides, 1994). The percent organic matter typically found in these sediments ranges between 6·0–13·0% (Table 1); high densities of macrophytes and seagrasses are clearly responsible for the high amounts of organic matter in these sediments (Hadjichristoforou & Argyrou, 1993b). The recent expansion of the macrophytic alga Caulerpa racemosa is likely to be related to non-point source inputs of nutrients; however, the relative importance of agriculture vs. sewage and other possible sources remain speculative at this time (Christodoulides,
114
T. S. Bianchi et al.
1994). Inherent differences in the productivity and life-history of this Red Sea migrant (C. racemosa), in relation to the native algal species of Cyprus, may also be an important factor controlling its distribution. Preliminary work has shown that the transport of nutrients in groundwaters to the sea is significant in certain regions (Christodoulides, 1994). If this is the case, it would explain how anthropogenic nutrient inputs are primarily being consumed by benthic primary production with little flux to the phytoplankton communities. Large-scale differences in Mediterranean waters Observations of higher chlorophyll a concentrations in deeper waters of the eastern Mediterranean as compared to the coastal waters of Cyprus may have resulted from the nutrient enhancement effects of warm-core eddies in this region (Krom et al., 1992, 1993). These quasi-stationary eddies are typically found south of Cyprus in the Levantine Basin and form as a result of the interaction between the mid-Mediterranean jet and the Erastothenes Seamount (Brenner et al., 1991; Krom et al., 1993). Much of the nutrient supply to the euphotic zone from deep upwelled waters in these eddies is believed to be derived from microbial regeneration of the previous year’s productivity (Krom et al., 1992). Despite these brief episodes of nutrient enhancement, the eastern Mediterranean remains more oligotrophic than the western Mediterranean principally due to the absence of inputs from nutrient-rich rivers such as the Rhone and Ebro (Coste et al., 1977). Moreover, it has been suggested that the removal of PO43" by dust particles rich in Fe " , derived from the Sahara Desert, may be an important mechanism for P removal in the water column of the south-eastern Mediterranean (Krom et al., 1991). The major differences between the oligotrophic regions of Mediterranean (eastern and western) and the open ocean are primarily the deep convective motions and mesoscale eddies that occur in the Mediterranean which result in significant annual shifts in nutrients and productivity (Millot, 1992).
Acknowledgements We would like to thank the crew of the RV Alkion and George Demetriou for their invaluable assistance in the collection of field samples. We are especially grateful to Dimitri Damaskinos for assisting with field sample collections and salinity determination. We also thank Christine Lambert for assisting in graphics and HPLC analysis. This work was supported by the U.S./Cyprus Fulbright Commission in Nicosia on a Fulbright Scholarship to T.S. Bianchi. References Appleby, P. G. & Oldfield, F. 1992 Application of 210Pb to sedimentation studies, In Uranium Series Disequilibrium, 2nd edition (Ivanovich, M. & Harmon, R. S., eds). Oxford Science Publications, Oxford, pp. 731–778. Azov, Y. 1986 Seasonal patterns of phytoplankton productivity and abundance in nearshore oligotrophic waters of the Levant Basin (Mediterranean). Journal of Plankton Research 8, 41–53. Bianchi, T. S. & Findlay, S. 1991 Decomposition of Hudson Estuary macrophytes: Photosynthetic pigment transformations and decay constants. Estuaries 14, 65–73. Bianchi, T. S., Findlay, S. & Dawson, R. 1993 Organic matter sources in the water column and sediments of the Hudson River estuary: the use of plant pigments as tracers. Estuarine, Coastal and Shelf Science 36, 359–376.
Plant pigments as biomarkers
115
Bianchi, T. S., Lambert, C. & Biggs, D. C. 1994 Distribution of chlorophyll a and phaeopigments in the northwestern Gulf of Mexico: a comparison between fluorimetric and high performance liquid chromatography measurements. Bulletin of Marine Science 56, 25–32. Bidigare, R. R., Frank, T. J., Zastrow, C. & Brooks, J. M. 1986 The distribution of algal chlorophylls and their degradation products in the southern Ocean. Deep Sea Research 33, 923–937. Brenner, S., Rosentraub, Z., Bishop, J. & Krom, M. 1991 The mixed layer thermocline cycle of a persistent warm core eddy in the Eastern Mediterranean. Dynamics of Atmospheres and Oceans 15, 457–477. Chisholm, S. W., Olsen, R. J., Zettler, E. R., Goericke, R., Waterbury, J. B. & Welschmeyer, N. A. 1988 A novel, free-living prochlorophyte abundant in the oceanic euphotic zone. Nature 334, 340–343. Christodoulides, A. 1994 Hydrogeological and hydrochemical survey of the coastal area between Liopetri River and Ayia Napa Village Cyprus in connection with the 1990–91 eutrophication phenomena in the sea. Ministry of Agriculture and Natural Resources, Department of Water Development Report, Cyprus. Coste, B., Jacques, G. & Minas, H. J. 1977 Sels nutritifs et production dans le Golfe du Lion et ses abords. Annals of the Institute of Oceanography 53, 189–202. Dickson, M. & Wheeler, P. A. 1993 Chlorophyll a concentrations in the North Pacific: Does a latitudinal gradient exist? Limnology and Oceanography 38, 1813–1818. Flynn, W. W. 1968 The determination of low levels of 210Po in environmental materials. Analytica Chimica Acta 43, 221–227. Hadjichristoforou, M. & Argyrou, M. 1993a Old Limassol Port area development project. Ministry of Agriculture and Natural Resources, Department of Fisheries Report, Cyprus. Hadjichristoforou, M. & Argyrou, M. 1993b List of marine plants recorded in coastal areas of Cyprus. Ministry of Agriculture and Natural Resources, Department of Fisheries Report, Cyprus. Hecht, A., Pinardi, N. & Robinson, A. R. 1988 Currents, water masses, eddies and jets in the Mediterranean Levantine Basin. Journal of Physical Oceanography 18, 1320–1353. Jacques, G., Minas, H. J., Minas, M. & Nival, P. 1973 Influence des conditions hivernales sur les productions phyto- et zooplanctoniques en Mediterranee Nord-Occidentale. 2 Biomasse et production phytoplanctonique. Marine Biology 23, 251–265. Klein, B. & Sournia, A. 1987 A daily study of the diatom spring bloom at Roscoff (France) in 1985. II. Phytoplankton pigment composition studied by HPLC analysis. Marine Ecology Progress Series 37, 265–275. Krom, M. D., Kress, N., Brenner, S. & Gordon, L. I. 1991 Phosphorus limitation of primary productivity in the eastern Mediterranean Sea. Limnology and Oceanography 36, 424–432. Krom, M. D., Brenner, S., Kress, N., Neori, A. & Gordon, L. I. 1992 Nutrient dynamics and new production in a warm-core eddy from the Eastern Mediterranean Sea. Deep Sea Research 39, 467–480. Krom, M. D., Brenner, S., Kress, N., Neori, A. & Gordon, L. I. 1993 Nutrient distributions during an annual cycle across a warm-core eddy from the E. Mediterranean Sea. Deep Sea Research 40, 805–825. Krishnaswami, S., Lal, E., Martin, J. M. & Meybeck, H. 1971 Geochronology of lake sediments. Earth and Planetary Science Letters 11, 407–414. Malanotte-Rizzoli, P. & Bergamasco, A. 1992 The baroclinic general circulation of the eastern Mediterranean Sea. Bulletin de I’Institut Oceanographique Monaco 11, 91–97. Malanotte-Rizzoli, P. & Hecht, A. 1988 Large-scale properties of the Eastern Mediterranean: a review. Oceanologica Acta 11, 323–335. Millot, C. 1992 Are there major differences between the largest Mediterranean seas? A preliminary investigation. Bulletin de I’Institut Oceanographique Monaco 11, 3–25. Ondrusek, M., Bidigare, R. R., Sweet, S. T., Defreitas, D. A. & Brooks, J. M. 1991 Distribution of phytoplankton pigments in the north Pacific Ocean in relation to physical and optical variability. Deep Sea Research 38, 243–266. Plante, R., Plante-Cuny, M. & Reys, J. 1986 Photosynthetic pigments of sandy sediments on the north Mediterranean coast: the spatial distribution and its effect on sampling strategies. Marine Ecology Progress Series 34, 133–141. Robinson, A. & Golnaraghi, M. 1992 Progress in the understanding of the eastern Mediterranean Sea. Bulletin de I’Institut Oceanographique Monaco 11, 65–73. Vaulot, D. & Partensky, F. 1992 Cell cycle distributions of prochlorophytes in the north western Mediterranean Sea. Deep Sea Research 39, 727–742. Vaulot, D., Partensky, F., Neveux, J., Mantoura, R. F. C. & Llewellyn, C. A. 1990 Winter presence of prochlorophytes in surface waters of the northwestern Mediterranean Sea. Limnology and Oceanography 35, 1156–1160. Wright, S. W., Jeffrey, S. W., Mantoura, R. F. C. et al. 1991 Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Marine Ecology Progress Series 77, 183–196.
Plant Pigments as Biomarkers of Organic Matter Sources in Sediments and Coastal Waters of Cyprus (eastern Mediterranean)
Thomas S. Bianchia, Andreas Demetropoulosb, Myroula Hadjichristophoroub, Marina Argyroub, M. Baskaranc, and Corey Lamberta a
Department of Ecology, Evolution and Organismal Biology, Tulane University, New Orleans, Louisiana 70118 U.S.A., bMinistry of Agriculture and Natural Resources, Department of Fisheries, 13 Aeolou Street, Nicosia, Cyprus, and c Department of Oceanography, Texas A&M University, Galveston, Texas 77553, U.S.A. Received 2 August 1994 and in revised form 10 October 1994
Keywords: plant pigments; radionuclides; sedimentation; Cyprus Plant pigments (chlorophylls and cartenoids) and radionuclides (226Ra and 210 Pb) were measured in the coastal waters and sediments off the coast of Cyprus (eastern Mediterranean), for the first time, in June and July 1993. Some of the lowest concentrations of chlorophyll a (10–90 ng l "1) were found in these highly oliogotrophic waters. Based on the presence of chlorophyll b and zeaxanthin, it appeared that chlorophytes, cyanobacteria, and prochlorophytes were the dominant phytoplankton classes. The phytoplankton assemblage off the coast of Cyprus was not very different when compared to the deeper regions of the eastern Mediterranean. However, there were significantly higher concentrations of pigments in sediments principally due to seagrasses and macroalgae. In fact the Red Sea migrant Caulerpa racemosa appears to be expanding its areal coverage along the coastline; it remains to be tested whether this is related to enhanced nutrient inputs or to inherent differences in the life-history characteristics of this migrant vs. native species. The very low sedimentation rates observed at all stations (0·38–0·78 mm year "1) further suggested that input from the water column to the sediments was minimal. The land-margin effects of Cyprus on the surrounding oligotrophic waters of the eastern Mediterranean appeared to only affect the composition and distribution of benthic macrophytes, with very little change in the phytoplankton assemblages. ? 1996 Academic Press Limited
Introduction In past years, much of the research using plant pigments as biomarkers of organic matter sources in the sediments and water column of the Mediterranean has centered on western regions (Plante et al., 1986; Klein & Sournia, 1987; Vaulot et al., 1990). Only recently have some measurements been made on phytoplankton biomass in the eastern Mediterranean; however, these studies have not used high performance liquid 0272–7714/96/010103+13 $12.00/0
? 1996 Academic Press Limited
104
T. S. Bianchi et al.
chromatography (HPLC) techniques and thus, have been restricted to measuring only chlorophyll a (Azov, 1986; Krom et al., 1993). While the western Mediterranean is also considered to be oligotrophic with low chlorophyll a values in summer ranging from 0·05 to 0·5 ìg l "1, many of the northern coastal margins experience massive phytoplankton blooms because of inputs from nutrient-rich rivers (Jacques et al., 1973; Klein & Sournia, 1987). Recent measurements in the south-eastern Mediterranean reveal that prevailing oligotrophic conditions result in low chlorophyll a concentrations ranging from 0·1 to 0·2 ìg l "1 (Krom et al., 1991). Changes in phytoplankton biomass and composition along land-margin ecosystems in this highly oligotrophic basin are likely to be very different than in the western Mediterranean. Cyprus is the easternmost Mediterranean island located in the Levantine basin of the eastern Mediterranean. The eastern Mediterranean is located east of the Sicilian Straights and comprises the Ionian and Levantine basins and the Adriatic and Aegean Seas. Compared with other regions of the Mediterranean, this is the most poorly studied, particularly the Levantine Basin. To date, much of the literature that exists on the eastern Mediterranean has centred on its physical oceanography [i.e. Physical Oceanography of the Eastern Mediterranean (POEM) project] (Hecht et al., 1988; Malanotte-Rizzoli & Hecht 1988; Millot 1992; Malanotte-Rizzoli & Bergamasco 1992; Robinson & Golnaraghi 1992). In fact, the Levantine intermediate water (LIW) formed in the basin, consists of dense salty water that is highly oligotrophic (Hecht et al., 1988). Other studies carried out in the deep waters of the Levantine basin have shown that primary productivity is limited by phosphorus (P) (Krom et al., 1991, 1992). Similarly, coastal waters off the coast of Israel also appear to be P-limited (Azov, 1986). Cyprus, the only large island situated in the eastern Levant, serves as an excellent site for examining land-margin interactions because of its isolation from significant riverine inputs. In this study, we used plant pigments as biomarkers to determine, for the first time, the dominant classes of phytoplankton in the coastal waters of Cyprus. We also used pigments to determine the dominant sources of carbon input to surficial sediments. Excess 210Pb concentrations in the sediments were also used to determine sedimentation rates. Methods and materials Study area and sampling Cyprus has an area of 9251 km2, the nearest distance to a coast is 75 km to southern Turkey, 102 km to Syria, and 384 km to Egypt. Water samples and sediments were collected for pigments aboard the RV Alkion at four regional stations along the coast of Cyprus in June and July 1993 (Figure 1). A shallow (25 m) and deep (50 m) station were sampled at each of the regional stations twice a month. Water samples for salinity and temperature profiles were taken at the deep stations using Nansen bottles. Surficial sediments were collected using an ‘ Orange Peel ’ grab sampler; sediment cores, for radionuclide analysis, were taken using a Piston-corer. Percent organic matter was measured by weight loss upon combustion at 500 )C for 24 h. Plant pigments Aliquots of 200–300 ml taken from the water sample were filtered through glass fiber filters (Whatman GF/F filters, 25 mm diameter, nominal pore size of 0·7 ìm) for plant pigment analysis (see Bianchi et al., 1993 for further details on extraction procedures).
Plant pigments as biomarkers
105
33°00'
Turkey
Greece Iráklion Rhodes Crete
Cyprus
Mediterranean Sea
35°30'
Syria Lebanon Beirut
km 100 0 100 200 300 400 Haifa Tel Aviv 100 200 Jordan Port Said Israel Alexandria
35°30'
miles100 50 0 Libya
35°00'
34°00'
Cyprus Famagusta
Latsi
35°00'
1 0 1 101520 km
Pafos Limassol
33°00'
5
0
10 15 20 miles
34°00'
Figure 1. Map of Cyprus showing the four regional stations sampled in June and July 1993. Although not shown in map there were two stations (25 m and 50 m depth) sampled at each regional station.
Due to time restrictions, we were not able to compare GF/F filters with 0·2 ìm pore size filters. Canthaxanthin was placed in all acetone extracts as an internal standard. Reverse-phase analysis was conducted using the method of Wright et al. (1991). A Waters Model 610 solvent delivery system was coupled with dual-channel detection using a Waters Model 996 photodiode array detector set at 438 nm for absorbance and a Milton–Roy fluorescence detector with excitation set at 440 nm and emission at >600 nm. The injector was connected via a guard column to a reverse-phase C18 Alltech adsorbosphere column (5 ìm particle size; 250 mm#4·6 mm i.d.). After injection (100 ìl) a gradient program (1 ml min "1) begins isocratically with mobile phase A (80:20 methanol: 0·5 M ammonium acetate, aq.; pH 7·2 v/v) which then ramps to 100% mobile phase B in 4 min (90:10 acetonitrile:HPLC grade water v/v) and then changes to 20% B and 80% mobile phase C (100% ethyl acetate) in 14 min. This is followed by a return to 100% B in 3 min with final ramping to 100% A in 3 min. This gradient was slightly modified for sediments resulting in a longer run time of 45 min. The method allowed for adequate resolution of dominant peaks of interest (Figure 2). High purity HPLC standards for chlorophylls a and b and á, â-carotenes were obtained from Sigma Co. Standards of the following carotenoids were kindly provided by Hoffman LaRoche Co., Basel, Switzerland: fucoxanthin; lutein; zeaxanthin; peridinin. Radionuclides Dried sediment core sections were pulverised using an agate mortar and pestle. Two grams of the powered samples were then transferred into microwave digestion vessels, to
106
T. S. Bianchi et al.
Relative absorbance
d
c
a
0
b
15.00 Retention time (min)
30.00
Figure 2. Absorbance chromatogram from a water sample collected at Famagusta (50 m) in June 1993. The identified pigment peaks are as follows: (a) Fucoxanthin; (b) Zeaxanthin; (c) Chlorophyll b; (d) Chlorophyll a.
which 10 ml of concentrated HF and 15 ml of concentrated HNO3 acids (trace metal grade) were added. A known amount of 209Po spike was added to the powder. Sediment digestions were accomplished using a commercial microwave digestion system (model CEMR -MD-81D). The contents in the digestion vessels were heated in the microwave at a maximum pressure of 90 p.s.i. for about 3 h. The solutions were then transferred to acid-cleaned teflon beakers containing 1 M HCL and taken to dryness on a hot plate. Polonium was electroplated onto silver planchets following the method of Flynn (1968), and then assayed for 210Pb by using an alpha spectrometer with a surface detector coupled to a Canberra S-100 multichannel analyser. The sediment samples were packed into 10-ml gamma counting vials and counted on a high purity Ge well-detector coupled to a Canberra S-100 multichannel analyser. The peak analysis of 226Ra (352 keV) was done using SPECTRAN-AT peak analysis software. The gamma counting equipment was calibrated with fly ash standards obtained from the Environmental Measurement Laboratory. Sedimentation rates were calculated using the methods described in Krishnaswami et al. (1971).
Plant pigments as biomarkers
107
Statistical analyses An Fmax was used prior to ANOVA analysis to check for homogeneity of variances. A two-way ANOVA was used to test for significant effects of station and depth on pigments. When ANOVA differences were significant, a Scheffe multiple range test was performed to detect for differences between means. Correlation analyses were performed using Pearsons product moment correlation matrix. Results Physicochemical data During June and July 1993, surface water temperatures (among all stations) ranged between 20–28 )C as compared to bottom temperatures which ranged between 16–20 )C. The salinity profiles were not highly stratified with the average salinity being maintained at c. 39. Transparency secchi disk readings ranged between 21–39 m with the highest clarity at the Latsi station. Water column pigments In June 1993, chlorophyll a concentrations ranged between 16–90 ng l "1 at all four regional stations, with a similar range of 10–90 ng l "1 in July 1993 (Figures 3–6). The highest concentrations occurred at the Pafos and Limassol stations, with the lowest measurable concentrations at Famagusta and Latsi. In June 1993, the subsurface chlorophyll a maxima generally occurred between 20–30 m at the 50-m stations. In July 1993, chlorophyll a concentrations were generally uniform throughout the water column with minor increases at depth at Famagusta and Latsi. Overall, the chlorophyll a concentrations in July, between 0–10 m, were significantly (P<0·05) lower than in June, except for Latsi. Chlorophyll b concentrations ranged between 0–76 ng l "1 in June 1993 and 2–55 ng l "1 in July 1993. Concentrations of the accessory pigment chlorophyll b, which is abundant in chlorophytes, were significantly correlated (P<0·05) with chlorophyll a concentrations at all stations in June and July. The highest concentrations (P<0·05) of chlorophyll b occurred at the Limassol station. Concentrations of fucoxanthin, a carotenoid commonly found in diatoms, ranged between 0–10 ng l "1 in June 1993 and only 0–3 ng l "1 in July 1993; the highest concentration (P<0·05) was found at the 50 m Pafos station. Zeaxanthin, a carotenoid found in cyanobacteria, had a concentration range of 0–14 ng l "1 in June 1993 and 0–16 ng l "1 in July 1993. Peridinin, a carotenoid marker for dinoflagellates, was generally below limits of detection in June 1993, however, it did reach detectable concentrations at stations Limassol and Pafos in July 1993 that ranged between 3–18 ng l "1 (data not shown). Concentrations of á-carotene were significantly higher (P<0·05) (3–31 ng l "1) than â-carotene (0–5 ng l "1) at all stations (data not shown). Sedimentary pigments Chlorophyll a concentrations in surface sediments (at all four stations) ranged between 0·05–0·46 ìg g dry sediment "1 and 0·03–0·37 ìg g dry sediment "1 in June and July 1993, respectively (Table 1). In July 1993, concentrations of chlorophyll a were always significantly (P<0·05) higher at the 25-m stations than at the 50-m stations, except for Famagusta. Concentrations of chlorophyll a were highest (P<0·05) at the Latsi and Famagusta stations in July 1993. Chlorophyll b concentrations ranged between 0·008–0·26 ìg g dry sediment "1 and 0·01–0·22 ìg g dry sediment "1 in June and July
108
T. S. Bianchi et al.
–1
–1
Pigment concentration (ng l ) 0
20
40
60
Pigment concentration (ng l ) 0
80 100 120
20
40
60
80 100 120
June 1993, 50 m 10
Depth (m)
Depth (m)
5
10
15
June 1993, 25 m
20
20
30
40
25
50
Pigment concentration (ng l–1) 0
20
40
60
Pigment concentration (ng l–1) 0
80 100 120
July 1993, 25 m
60
80 100 120
10
Depth (m)
Depth (m)
15
40
July 1993, 50 m
5
10
20
20
30
20
40
25
50
Figure 3. Water column pigment profiles (ng l "1) at the Famagusta station (25 m and 50 m) off the coast of Cyprus in June and July 1993. -, Chlorophyll a; ., Chlorophyll b; 4, Fucoxanthin; :, Zeaxanthin.
1993, respectively. Fucoxanthin and zeaxanthin concentrations were generally low with concentrations ranging between 0·01–0·26 ìg g dry sediment "1 and 0·01– 0·16 ìg g dry sediment "1 in June and July 1993, respectively. Lutein, a carotenoid commonly found in chlorophytes, ranged between 0·02–0·32 ìg g dry sediment "1 with highest concentration occurring at the Limassol station (data not shown). Sedimentation rates There appeared to be no sediment mixing at all four stations, the excess 210Pb penetration depths varied between 5–8 cm. The linear sedimentation rates at the four
Plant pigments as biomarkers
109
–1
–1
Pigment concentration (ng l ) 0
20
40
60
Pigment concentration (ng l ) 0
80 100 120
20
June 1993, 25 m
Depth (m)
Depth (m)
80 100 120
10
10
15
20
30
20
40
25
50
Pigment concentration (ng l–1) 20
40
60
Pigment concentration (ng l–1) 0
80 100 120
July 1993, 25 m
15
40
60
80 100 120
10
Depth (m)
10
20
July 1993, 50 m
5
Depth (m)
60
June 1993, 50 m
5
0
40
20
30
20
40
25
50
Figure 4. Water column pigment profiles (ng l "1) at the Limassol station (25 m and 50 m) off the coast of Cyprus in June and July 1993. -, Chlorophyll a; ., Chlorophyll b; 4, Fucoxanthin; :, Zeaxanthin.
stations varied between 0·38–0·70 mm year "1. The parent-supported 226Ra concentration varied between 0·40–0·76 dpm g "1. The linear sedimentation rates at Latsi and Pafos were comparable, 0 38 mm year "1 and 0·44 mm year "1, respectively. The fastest sedimentation rate was observed at Limassol, 0·70 mm year "1, the sedimentation rate at Famagusta was 0·58 mm year "1. Discussion Organic matter sources in the water column Chlorophyll a concentrations (commonly used to estimate primary production biomass) in the surface waters off the coast of Cyprus are among the lowest values (10–90 ng l "1)
110
T. S. Bianchi et al.
–1
–1
Pigment concentration (ng l ) 0
20
40
60
Pigment concentration (ng l ) 0
80 100 120
40
60
80 100 120
10
10
15
June 1993, 25 m
Depth (m)
Depth (m)
5
20
20
20
30
40 June 1993, 50 m
25
50
Pigment concentration (ng l–1) 0
20
40
60
Pigment concentration (ng l–1) 0
80 100 120
July 1993, 25 m
60
80 100 120
10
Depth (m)
Depth (m)
15
40
July 1993, 50 m
5
10
20
20
30
20
40
25
50
Figure 5. Water column pigment profiles (ng l "1) at the Pafos station (25 m and 50 m) off the coast of Cyprus in June and July 1993. -, Chlorophyll a; ., Chlorophyll b; 4, Fucoxanthin; :, Zeaxanthin.
ever observed in nearshore waters. The highest concentrations of chlorophyll a, which occurred at the Pafos and Limassol stations, are still remarkably lower than the range of concentrations (0·15–0·25 ìg l "1) observed in deeper oligotrophic waters of the eastern Mediterranean (Krom et al., 1991). The highest chlorophyll a inventories also occurred at Limassol and Pafos (50-m stations) in June 1993; the lowest occurred at Pafos (25-m station) in July 1993 (Table 2). The highest chlorophyll a inventory at Limassol (2·17 mg m "2) may be related to a greater input of anthropogenic nutrients (i.e. agriculture, sewage and industry) at this station, which is the largest coastal city in Cyprus. In a recent study of the coastal aquifer, between Limassol and Famagusta, it was concluded that fertilizers used in excess by farmers (primarily for potatoes and citrus)
Plant pigments as biomarkers
111
–1
–1
Pigment concentration (ng l ) 0
20
40
60
Pigment concentration (ng l ) 0
80 100 120
20
10
15
20
30
20
40
25
50
Pigment concentration (ng l–1) 20
40
60
Pigment concentration (ng l–1) 0
80 100 120
July 1993, 25 m
40
60
80 100 120
10
Depth (m)
Depth (m)
20
July 1993, 50 m
5
15
80 100 120
10
Depth (m)
Depth (m)
5
10
60
June 1993, 50 m
June 1993, 25 m
0
40
20
30
20
40
25
50
Figure 6. Water column pigment profiles (ng l "1) at the Latsi station (25 m and 50 m) off the coast of Cyprus in June and July 1993. -, Chlorophyll a; ., Chlorophyll b; 4, Fucoxanthin; :, Zeaxanthin.
are clearly contributing to the developing eutrophication problems along the coast (Christodoulides, 1994). For example, based on a total groundwater outflow of 1000 m3 day "1 for this region, the total annual nutrient outflows of nitrate and phosphorous to the sea are 20 metric tons and 50 kg, respectively (Christodoulides, 1994). The significantly lower chlorophyll a concentrations observed in July (between 0–10 m) are probably due to the result of changes in nutrients, rather than grazing effects because of the very low phaeopigment concentrations observed at all stations: chlorophyllides ranged between 0·05–0·78 ng l "1, total phaeophytins ranged between 0·12–1·12 ng l "1, and total phaeophorbides ranged between 1·14–5·13 ng l "1.
112
T. S. Bianchi et al.
T 1. Percent organic matter and plant pigment concentrations (ìg g "1 dry sediment "1) in surficial sediments (0–2 cm) collected from four locations at 25 m and 50 m depths, in July 1993 (only 50 m depth for June 1993) off the coast of Cyprus in the eastern Mediterranean, SE= &.
Date June 1993 June 1993 June 1993 June 1993 July 1993 July 1993 July 1993 July 1993 July 1993 July 1993 July 1993 July 1993
Station/depth
% Organic matter
Famagusta (50 m) Limassol (50–m) Pafos (50 m) Latsi (50 m) Famagusta (25 m) Famagusta (50 m) Limassol (25 m) Limassol (50 m) Pafos (25 m) Pafos (50 m) Latsi (25 m) Latsi (50 m)
9·7&1·9 9·6&1·0 10·1&2·1 13·2&3·1 9·7&1·4 10·3&1·1 6·8&2·2 9·3&2·1 6·8&2·6 7·4&1·9 6·2&1·3 9·7&2·7
Chlorophyll a
Chlorophyll b (ìg g
0·46&0·12 0·05&0·01 0 12&0·03 0·09&0·01 0·28&0·01 0·34&0·05 0·23&0·02 0·12&0·002 0·19&0·003 0·05&0·004 0·37&0·03 0·03&0·003
"1
Fucoxanthin
dry sediment
0·09&0·01 0·26&0·001 0·05&0·004 0·008&0·002 0·04&0·003 0·07&0·03 0·11&0·001 0·02&0·001 0·22&0·001 0·01&0·001 0·07&0·01 0·01&0·001
Zeaxanthin
"1
)
0·03&0·001 0·01&0·001 0·26&0·01 0·03&0·01 0·11&0·002 0·18&0·03 0·14&0·004 0·08&0·01 0·08&0·01 0·03&0·01 0·21&0·004 0·13&0·002
0·05&0·01 0·16&0·007 0·01&0·002 0·01&0 002 0·03&0·001 0·07&0·02 0·07&0·02 0·03&0·01 0·01&0·001 0·02&0·001 0·03&0·003 0·01&0·001
T 2. Chlorophyll a inventories at four stations in the coastal waters of Cyprus in June and July 1993
Date
Station/depth (m)
Chlorophyll a inventory (mg m "2)
June 1993 June 1993 June 1993 June 1993 June 1993 June 1993 June 1993 June 1993 July 1993 July 1993 July 1993 July 1993 July 1993 July 1993 July 1993 July 1993
Famagusta (25 m) Famagusta (50 m) Limassol (25 m) Limassol (50 m) Pafos (25 m) Pafos (50 m) Latsi (25 m) Latsi (50 m) Famagusta (25 m) Famagusta (50 m) Limassol (25 m) Limassol (50 m) Pafos (25 m) Pafos (50 m) Latsi (25 m) Latsi (50 m)
1·21 1·02 1·48 2·17 1·29 2·05 0·89 1·76 0·66 1·21 0·63 0·78 0·38 0·93 0·44 1·00
Based on relatively higher concentrations of the accessory pigments zeaxanthin and chlorophyll b, it appears that most of the phytoplankton in the coastal waters of Cyprus is comprised of chlorophytes, cyanobacteria, and prochlorophytes. These classes of phytoplankton typically dominate in oligotrophic oceanic waters (Chisholm et al., 1988; Ondrusek et al., 1991). While chlorophyll b is a marker for chlorophytes and zeaxanthin is commonly found in cyanobacteria, prochlorophytes have been found to contain both of these pigments (Bidigare et al., 1986; Chisholm et al., 1988; Ondrusek et al., 1991). Thus, the presence of prochlorophytes makes it very difficult to separate the relative importance of chlorophytes vs. cyanobacteria. Prochlorophytes are prokaryotic picoplankton commonly found in oligotrophic oceanic waters; they are characterized as
Plant pigments as biomarkers
113
having chlorophyll b, divinyl chlorophyll a (substituted for chlorophyll a), zeaxanthin, and á-carotene (substituted for â-carotene) (Chisholm et al., 1988). Recent work has shown that prochlorophytes are not restricted to oceanic regimes and have been found to be important in the north-western Mediterranean as well as the Gulf of Mexico (Vaulot et al., 1990; Vaulot & Partensky, 1992; Bianchi et al., 1994). The typical chlorophyll a/zeaxanthin ratio of prochlorophytes is c. 3·0; the range of ratios found at the Cyprus stations (at the 50-m station in the upper 10 m) was between 1·05–15·93 (mean&6·18 SD&4·1). Except for a few high ratios at Latsi and Pafos, most of the ratios fall within the range of 2–6, very close to that of cyanobacteria and prochlorophytes. The chlorophyll a/zeaxanthin ratios, at the 25-m station and in the lower water column at both depths, tend to be higher presumably due to detrital macrophytic inputs from resuspended bottom materials. The ratios of older macrophytic detritus are higher due to differences in the decay kinetics of chlorophyll a and zeaxanthin, since chlorophyll a decays more rapidly (Bianchi & Findlay, 1991). The reverse-phase HPLC method used here does not resolve divinyl chlorophyll a, thus, we were not able to determine the relative importance of prochlorophytes. However, the dominance of á-carotene vs. â-carotene in these waters further suggests that prochlorophytes are likely to be a component of the phytoplankton. The presence of fucoxanthin in very low concentrations suggests that diatoms contributed only marginally to the total phytoplankton assemblage in June and July 1993. If prochlorophytes are a significant component of the phytoplankton assemblage, using GF/F filters would likely result in an underestimation of chlorophyll a concentrations because of loss through the pores; recent studies show that 0·2 ìm pore size filters can result in significant differences (Dickson & Wheeler, 1993; Bianchi et al., 1994). Organic matter sources in sediments Concentrations of chlorophyll a in sediments along the coast of Cyprus were always found to be substantially higher than those found in the water column—due to high densities of macroalgae and seagrasses. The very low sedimentation rates observed at all stations further suggests that there is low input from the water column to the sediments. The range of sedimentation rates observed here (0·38–0·70 mm year "1) are considerably lower than that found in most coastal regions (Appleby & Oldfield, 1992). The high incidence of light to these sediments (estimated by secchi disk), at both the 25-m and 50-m stations, should allow for rapid expansion phytobenthos—if pulsed with nutrients. The highest chlorophyll a concentrations in sediments occurred at Famagusta and Latsi, however, the density of macrophytes in these regions may not be significantly greater than the other stations. This inconsistency can likely be explained by high spatial heterogeneity commonly found within these benthic communities (Hadjichristoforou & Argyrou, 1993a). The availability of nutrients to these macrophytes will also be driven in part by the regeneration of nutrients from decaying macrophyte detritus. However, it is likely that anthropogenic inputs from agriculture and tourism may be more of a controlling factor (Christodoulides, 1994). The percent organic matter typically found in these sediments ranges between 6·0–13·0% (Table 1); high densities of macrophytes and seagrasses are clearly responsible for the high amounts of organic matter in these sediments (Hadjichristoforou & Argyrou, 1993b). The recent expansion of the macrophytic alga Caulerpa racemosa is likely to be related to non-point source inputs of nutrients; however, the relative importance of agriculture vs. sewage and other possible sources remain speculative at this time (Christodoulides,
114
T. S. Bianchi et al.
1994). Inherent differences in the productivity and life-history of this Red Sea migrant (C. racemosa), in relation to the native algal species of Cyprus, may also be an important factor controlling its distribution. Preliminary work has shown that the transport of nutrients in groundwaters to the sea is significant in certain regions (Christodoulides, 1994). If this is the case, it would explain how anthropogenic nutrient inputs are primarily being consumed by benthic primary production with little flux to the phytoplankton communities. Large-scale differences in Mediterranean waters Observations of higher chlorophyll a concentrations in deeper waters of the eastern Mediterranean as compared to the coastal waters of Cyprus may have resulted from the nutrient enhancement effects of warm-core eddies in this region (Krom et al., 1992, 1993). These quasi-stationary eddies are typically found south of Cyprus in the Levantine Basin and form as a result of the interaction between the mid-Mediterranean jet and the Erastothenes Seamount (Brenner et al., 1991; Krom et al., 1993). Much of the nutrient supply to the euphotic zone from deep upwelled waters in these eddies is believed to be derived from microbial regeneration of the previous year’s productivity (Krom et al., 1992). Despite these brief episodes of nutrient enhancement, the eastern Mediterranean remains more oligotrophic than the western Mediterranean principally due to the absence of inputs from nutrient-rich rivers such as the Rhone and Ebro (Coste et al., 1977). Moreover, it has been suggested that the removal of PO43" by dust particles rich in Fe " , derived from the Sahara Desert, may be an important mechanism for P removal in the water column of the south-eastern Mediterranean (Krom et al., 1991). The major differences between the oligotrophic regions of Mediterranean (eastern and western) and the open ocean are primarily the deep convective motions and mesoscale eddies that occur in the Mediterranean which result in significant annual shifts in nutrients and productivity (Millot, 1992).
Acknowledgements We would like to thank the crew of the RV Alkion and George Demetriou for their invaluable assistance in the collection of field samples. We are especially grateful to Dimitri Damaskinos for assisting with field sample collections and salinity determination. We also thank Christine Lambert for assisting in graphics and HPLC analysis. This work was supported by the U.S./Cyprus Fulbright Commission in Nicosia on a Fulbright Scholarship to T.S. Bianchi. References Appleby, P. G. & Oldfield, F. 1992 Application of 210Pb to sedimentation studies, In Uranium Series Disequilibrium, 2nd edition (Ivanovich, M. & Harmon, R. S., eds). Oxford Science Publications, Oxford, pp. 731–778. Azov, Y. 1986 Seasonal patterns of phytoplankton productivity and abundance in nearshore oligotrophic waters of the Levant Basin (Mediterranean). Journal of Plankton Research 8, 41–53. Bianchi, T. S. & Findlay, S. 1991 Decomposition of Hudson Estuary macrophytes: Photosynthetic pigment transformations and decay constants. Estuaries 14, 65–73. Bianchi, T. S., Findlay, S. & Dawson, R. 1993 Organic matter sources in the water column and sediments of the Hudson River estuary: the use of plant pigments as tracers. Estuarine, Coastal and Shelf Science 36, 359–376.
Plant pigments as biomarkers
115
Bianchi, T. S., Lambert, C. & Biggs, D. C. 1994 Distribution of chlorophyll a and phaeopigments in the northwestern Gulf of Mexico: a comparison between fluorimetric and high performance liquid chromatography measurements. Bulletin of Marine Science 56, 25–32. Bidigare, R. R., Frank, T. J., Zastrow, C. & Brooks, J. M. 1986 The distribution of algal chlorophylls and their degradation products in the southern Ocean. Deep Sea Research 33, 923–937. Brenner, S., Rosentraub, Z., Bishop, J. & Krom, M. 1991 The mixed layer thermocline cycle of a persistent warm core eddy in the Eastern Mediterranean. Dynamics of Atmospheres and Oceans 15, 457–477. Chisholm, S. W., Olsen, R. J., Zettler, E. R., Goericke, R., Waterbury, J. B. & Welschmeyer, N. A. 1988 A novel, free-living prochlorophyte abundant in the oceanic euphotic zone. Nature 334, 340–343. Christodoulides, A. 1994 Hydrogeological and hydrochemical survey of the coastal area between Liopetri River and Ayia Napa Village Cyprus in connection with the 1990–91 eutrophication phenomena in the sea. Ministry of Agriculture and Natural Resources, Department of Water Development Report, Cyprus. Coste, B., Jacques, G. & Minas, H. J. 1977 Sels nutritifs et production dans le Golfe du Lion et ses abords. Annals of the Institute of Oceanography 53, 189–202. Dickson, M. & Wheeler, P. A. 1993 Chlorophyll a concentrations in the North Pacific: Does a latitudinal gradient exist? Limnology and Oceanography 38, 1813–1818. Flynn, W. W. 1968 The determination of low levels of 210Po in environmental materials. Analytica Chimica Acta 43, 221–227. Hadjichristoforou, M. & Argyrou, M. 1993a Old Limassol Port area development project. Ministry of Agriculture and Natural Resources, Department of Fisheries Report, Cyprus. Hadjichristoforou, M. & Argyrou, M. 1993b List of marine plants recorded in coastal areas of Cyprus. Ministry of Agriculture and Natural Resources, Department of Fisheries Report, Cyprus. Hecht, A., Pinardi, N. & Robinson, A. R. 1988 Currents, water masses, eddies and jets in the Mediterranean Levantine Basin. Journal of Physical Oceanography 18, 1320–1353. Jacques, G., Minas, H. J., Minas, M. & Nival, P. 1973 Influence des conditions hivernales sur les productions phyto- et zooplanctoniques en Mediterranee Nord-Occidentale. 2 Biomasse et production phytoplanctonique. Marine Biology 23, 251–265. Klein, B. & Sournia, A. 1987 A daily study of the diatom spring bloom at Roscoff (France) in 1985. II. Phytoplankton pigment composition studied by HPLC analysis. Marine Ecology Progress Series 37, 265–275. Krom, M. D., Kress, N., Brenner, S. & Gordon, L. I. 1991 Phosphorus limitation of primary productivity in the eastern Mediterranean Sea. Limnology and Oceanography 36, 424–432. Krom, M. D., Brenner, S., Kress, N., Neori, A. & Gordon, L. I. 1992 Nutrient dynamics and new production in a warm-core eddy from the Eastern Mediterranean Sea. Deep Sea Research 39, 467–480. Krom, M. D., Brenner, S., Kress, N., Neori, A. & Gordon, L. I. 1993 Nutrient distributions during an annual cycle across a warm-core eddy from the E. Mediterranean Sea. Deep Sea Research 40, 805–825. Krishnaswami, S., Lal, E., Martin, J. M. & Meybeck, H. 1971 Geochronology of lake sediments. Earth and Planetary Science Letters 11, 407–414. Malanotte-Rizzoli, P. & Bergamasco, A. 1992 The baroclinic general circulation of the eastern Mediterranean Sea. Bulletin de I’Institut Oceanographique Monaco 11, 91–97. Malanotte-Rizzoli, P. & Hecht, A. 1988 Large-scale properties of the Eastern Mediterranean: a review. Oceanologica Acta 11, 323–335. Millot, C. 1992 Are there major differences between the largest Mediterranean seas? A preliminary investigation. Bulletin de I’Institut Oceanographique Monaco 11, 3–25. Ondrusek, M., Bidigare, R. R., Sweet, S. T., Defreitas, D. A. & Brooks, J. M. 1991 Distribution of phytoplankton pigments in the north Pacific Ocean in relation to physical and optical variability. Deep Sea Research 38, 243–266. Plante, R., Plante-Cuny, M. & Reys, J. 1986 Photosynthetic pigments of sandy sediments on the north Mediterranean coast: the spatial distribution and its effect on sampling strategies. Marine Ecology Progress Series 34, 133–141. Robinson, A. & Golnaraghi, M. 1992 Progress in the understanding of the eastern Mediterranean Sea. Bulletin de I’Institut Oceanographique Monaco 11, 65–73. Vaulot, D. & Partensky, F. 1992 Cell cycle distributions of prochlorophytes in the north western Mediterranean Sea. Deep Sea Research 39, 727–742. Vaulot, D., Partensky, F., Neveux, J., Mantoura, R. F. C. & Llewellyn, C. A. 1990 Winter presence of prochlorophytes in surface waters of the northwestern Mediterranean Sea. Limnology and Oceanography 35, 1156–1160. Wright, S. W., Jeffrey, S. W., Mantoura, R. F. C. et al. 1991 Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Marine Ecology Progress Series 77, 183–196.