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Sediment System of an Eastern Mediterranean Coastal Area
Estuarine, Coastal and Shelf Science (2002) 55, 415–426 doi:10.1006/ecss.2001.0915, available online at http://www.idealibrary.com on
Seasonality of Algal Pigments in the Sea Water and Interstitial Water/Sediment System of an Eastern Mediterranean Coastal Area A. Metaxatos and L. Ignatiades NCSR ‘ Demokritos ’, Institute of Biology, Aghia Paraskevi, 15310 Attiki, Greece Received 20 February 2001 and accepted in revised form 1 October 2001 An effort has been made herein to describe the seasonal variation of the microalgal pigments, nutrient concentrations and other physicochemical parameters in a sandy and eutrophic coastal area of North Euboikos gulf, Aegean Sea. The algal pigments chlorophyll a, chlorophyll b, chlorophyll c, fucoxanthin, zeaxanthin, peridinin, 19 -hexanoyloxyfucoxanthin, 19 -butanoyloxyfucoxanthin, pheophytin a and pheophytin b were analysed by High Performance Liquid Chromatography (HPLC) in the interstitial water/sediment system (IW) and sea water (SW) during 1999. Chl a was recorded in all samples and its annual mean values were 1709 mg m 3 in IW and 0·76 mg m 3 in SW. Fucoxanthin was the most abundant accessory pigment in both media giving a total average 1183 mg m 3 in IW and 0·49 mg m 3 in SW while the pheophytin a mean values were 1183 mg m 3 in IW and 0·49 mg m 3 in SW. The vertical distributions of various pigments in the three layers of IW (0–3 mm, 10–13 mm and 30–33 mm) fluctuated quantitatively on a monthly basis. Only in a few cases certain pigments (Chl a, Chl c, Fuco and HEX) were equally distributed in the three layers (Chl c: June–July, Fuco: January–December, HEX: March, Chl a: April–May–January), while the distribution between layers was unequal during the rest of the year. Special emphasis was given in the analysis of data derived from the top layer (0–3 mm) of interstitial water/sediment system (IW) and near bottom water (NBW) e.g. the interface of two media. The ratios of pigment/Chl a and correlations were also estimated in order to enlighten the relationships among various pigments spatially and temporally. By using stepwise forward multiple regression we determined the pigments (Fuco, HEX, Chl c and BUF) that are the significant contributors collectively to the total Chl a biomass and we revealed differences in pigment contributions among various layers of IW or depths in SW and between media. 2002 Elsevier Science Ltd. All rights reserved.
Keywords: algal pigments; HPLC; interstitial water/sediment system; Euboikos gulf; Aegean Sea
Introduction The sea bottom environment of inshore waters and its relationship with the water column above it has been the subject of numerous studies. These entities are not completely independent, since the water contributes by exchange to the interstitial water (water content of the sediment) and receives material from it (Moore, 1962). In permeable beds advective interfacial water can flow through them providing a fast carrier for the exchange of substances between the water column and the upper sediment layers, so that permeable shelf sands represent gigantic filter systems (Huettel & Rusch, 2000). Thus, in shelf environments, up to 50% of the primary production can settle through the water column and most of this particulate organic material is mineralized in the sediment, and its decomposition products are returned to the water column (Huettel et al., 1996). Benthic microalgal communities include both motile algae (epipelic diatoms, cyanobacteria, 0272–7714/02/090415+12 $35.00/0
flagellates, etc.) as well as sessile algae attached to the sand grains (epipsammic diatoms, sporelings) which build up an important autotrophic biomass for the ecosystem, with values of up to 560 mg m 2 in the upper centimetre of sediments (Wolfstein et al., 2000). These communities are also involved in many fundamental processes like the production of labile matter, the regeneration of nutrients and the regulation of oxygen and they constitute an important source of food for heterotrophic mollusca like suspension-feeding bivalves (Bodoy & Plante-Cuny, 1984; Bayne et al., 1987; Jørgensen, 1990; Hawkins & Bayne, 1992). Their contribution to the carbon budgets of shallow-water systems has been extensively documented (Varela & Penas, 1985). Due to the relevance of benthic primary producers to the ecosystem, estimating their abundance in sea water as well as in the upper centimetre of sediment and its interstitial water altogether (or the interstitial water/sediment system) remains an important goal. In the literature, information on the pigment content of 2002 Elsevier Science Ltd. All rights reserved.
416 A. Metaxatos & L. Ignatiades N
39 Eu
boi
38 ge
37
an Se
Euripus Canal
a
s S1 S2
Ae
ko
36 35
21 22 23 24 25 26 27 28
Saronicos Gulf
Aegean Sea
F 1. Map of the study area in North Euboikos gulf, Aegean Sea.
sediments are mostly referred to Chl a determinations or its degradation products (Sun et al., 1991, 1994; MacIntyre & Cullen, 1995; Duineveld et al., 2000) whereas studies on other major or accessory pigments are scanty (Abele, 1991; Levinton & McCartney, 1991; Lucas & Holligan, 1999). Also, compared to the great number of results published worldwide, information on pigment content of the sediments in the Mediterranean Sea is poor and limited to the western areas (Rodriguez & Guerrero, 1994; Barlow et al., 1995) whereas information from the eastern coastlines of this oligotrophic sea is scant (Bianchi et al., 1996; Duineveld et al., 2000). The main objective of this study was to present a complete description of the dominant and accessory pigments (chlorophyll a, chlorophyll b and chlorophyll c, fucoxanthin, zeaxanthin, peridinin, 19 -hexanoyloxyfucoxanthin, 19 -butanoyloxyfucoxanthin, pheophytin a and pheophytin b) in sea water and in the interstitial water/sediment system of Euboikos gulf, Aegean Sea (Eastern Mediterranean). This inshore environment, although it is occupied by large stocks of bivalves, is unexplored regarding the important parameters such as phytoplankton biomass, grain size distribution and the related physicochemical conditions that might affect infaunal populations. Material and methods Study site North Euboikos gulf (Aegean Sea) extends along the western coast of Evia island, which is separated from
the Greek mainland by the Euripus channel that is 40 m wide, 60 m long and 8 m deep. Two inshore stations 350 m apart were selected: S1 having maximum depth of 3·5 m and S2 having maximum depth of 8 m (Figure 1). The benthic community in this coastal area is dominated by bivalves (Callista chione, Venus verrucosa, Loripes lacteus, Loripes fragilis, Hiatella arctica, Dosinia exoleta, Acanthocardia sp., Parvicardium sp. etc.). The stations represent the limits of C. chione’s geographical distribution, which is the most abundant bivalve regarding the biomass (dry weight: 154·3 gr m2) with an average population density of 6 individuals m2 (data in preparation).
Water sampling and analysis Sea water samples (SW) were collected monthly during 1999 with a Hydro-bios sampler equipped with an inverted thermometer from two depths (a) surface sea water (SSW: 0·3 m below the surface) and (b) near bottom water (NBW: 0·3 m above the seabed). Vertical profiles of temperature (inverted thermometer), salinity and dissolved oxygen were measured in situ with portable apparatus (LaMotte, Portable Electronic Lab., model DCL-05). Samples for the nutrient analyses were stored in 150 ml polyethylene bottles and kept frozen at 30 C until analysis of phosphates, nitrates, nitrites, silicates (Strickland & Parsons, 1974) and ammonia (Liddicoat et al., 1976). Samples for phytopigment analysis were kept at 4 C in the dark and filtered through millipore
Algal pigments of an Eastern Mediterranean coastal area 417
filters (0·2 m pore size). The filters were stored in aluminium foil and kept at 90 C until further analysis by reverse-phase HPLC (model HP 1100 series) with a column Spherisorb ODS2 25 cm4·6 mm ID, 5 m particle size with a loop 50 l. Pigment detection was at 440 nm and the solvent system, flow rate and gradient system was according to Wright et al. (1991). Pigments were extracted from thawed filters using 0·5 ml 90% acetone at 4 C in the dark for several hours. For ease of comparison pigment concentrations are reported in mg m 3 in both media e.g. mg of pigments in one cubic metre of SW or mg of pigments in one cubic metre of IW (grains and interstitial water). Samples for estimation of total suspended solids (TSS) and ash free dry weight (AFDW) in sea water were kept at 4 C in the dark. A well-mixed subsample was filtered through a weighed standard glass-fibre filter and the residue retained on the filter was dried to a constant weight at 75 C. The increase in weight of the filter represented the total suspended solids (APHA et al., 1992). Afterwards, the residue was ignited to constant weight at 50050 C and the remaining solids represented the ash-weight while the weight lost on ignition represented the ash-free dry weight which is a rough approximation of the amount of organic solids (APHA et al., 1992; Botto & Iribarne, 2000). Sediment sampling Sediment for interstitial water/sediment (IW) analyses was collected by divers in 3–5 acrylic syringes (32 mm inner diameter) that were pushed into the sandy bottom, sealed in situ (for retaining both sediment and its interstitial water) and frozen in liquid nitrogen on board. To avoid mixing of the layers during transportation to the laboratory the samples were kept perpendicular. The cores were sectioned in 3 mm slices with a methanol-rinsed razor blade and care was taken to ensure that the sediment surface was perpendicular to the long axis of the cores to minimize unevenness in the thickness of the surficial layer (MacIntyre & Cullen, 1995). The slices (0–3 mm, 10–13 mm and 30–33 mm) were kept at 90 C until further analysis. A series of experiments was also performed in trays used in experimental bivalve aquaculture in order to evaluate the entire population of algae derived from the water column or resuspension from the seabed. Thus, in May 1999 a total of 35 plastic trays (30 cm20 cm13 cm) were filled with defaunated sand and secured on the surface of the seabed of the two stations S1 and S2 by two metal pegs anchoring in
the deep sediment. To prevent sediment loss during diving operations the trays were sealed in plastic bags which were later removed by divers on the sea bottom. The first tray sampling was in June 1999 and subsequent samplings were carried out monthly for 6 months thereafter. No sampling was carried out in December 1999 due to a storm after which all experimental trays were lost. Defaunated sand was obtained by air drying the sediment under shade for 1 month. Before use, this air-dried sand was examined under a microscope to ensure that there were no live macroinfauna, and it was also analysed for pigments. HPLC pigment analysis and sediment analysis HPLC pigment analysis was used to study the quantitative and qualitative pigment identification in the SW (sea water) and IW (interstitial water/ sediment system). The followed protocol (Wright et al., 1991) combined advantages of earlier methods and it was recommended by the SCOR Working group 78 (Wright & Jeffrey, 1997). This standard protocol makes use of the retention capacity of ammonium acetate-containing mobile phase and the special selectivity of acetonitrile-based eluents for carotenoids separation (Zapata et al., 2000). We used acetone instead of methanol as extraction solvent because it yielded significantly better recoveries of pheopigments while methanol as an extraction solvent may underestimate pheopigment concentrations as well as Chl a concentrations in benthic field samples (Buffan-Dubau & Carman, 2000). The slices of frozen interstitial water/sediment were added in equal volume to 90% acetone (sediment: acetone 90%, v/v, 1/1) for a direct extraction of pigments from the interstitial water contained in the sediment pores and most likely pigments attached to the surface of grains. This mixture was stirred and vortexed several times before the liquid phase was recovered from IW by centrifugation (5 min, 720g). The supernatant was kept at +4 C in the dark for several hours to complete pigment extraction and then it was centrifuged again to clarify the extract under the same conditions (ultrasonic bath was omitted to avoid pigment degradation). The final supernatant was injected into the HPLC system and detected at 440 nm (Wright et al., 1991). The extraction protocol presented here is similar to MacIntyre and Cullen (1995) and Light and Beardall (1998). Calibration was done with external standards obtained by the Sigma Chem. Co, and the International Agency for 14C determination, Denmark, except for pheophytin a and pheophytin b.
418 A. Metaxatos & L. Ignatiades T 1. The percentage distribution of grain diameters (Phi) in both stations (S1, S2) and trays Diameter of grain (mm)
Phi
S1 % meanSD
S2 % meanSD
Trays % meanSD
>2 >1 >0·5 >0·25 >0·125 >0·063 <0·063
1 0 1 2 3 4 >4
14·766·98 20·287·96 26·823·83 27·109·07 9·915·12 0·760·36 0·210·12
13·808·67 12·876·18 24·946·49 36·6213·72 10·695·86 0·830·43 0·260·24
19·347·50 31·201·97 25·914·06 17·514·03 5·451·17 0·390·09 0·140·04
T 2. The annual mean values and ranges of nutrients, temperature, salinity. Total suspended solids (TSS) and ash free dry weight (AFDW) in the two stations Nutrients (g-at l 1) Stations
N-NO2
N-NO3
N-NH4
P-PO4
SiO2
S1 S2 Ranges
0·110·10 0·110·10 0·02–0·402
1·321·30 1·321·38 0·07–6·1
1·351·68 1·331·77 0·22–6·19
0·390·90 0·360·96 0·04–4·1
4·691·92 4·841·90 1·46–7·96
Stations
T C
Salinity PSU
TSS (mg l 1)
AFDW (mg l 1
S1 S2 Ranges
20·506·6 20·506·5 11·1–29·6
37·500·70 37·500·90 36·8–38·2
0·0260·014 0·0230·009 0·0150·026
0·0180·12 0·0190·012 0·0130·021
These pigments were prepared in the laboratory by acidification (1 M HCl) of the corresponding chlorophylls and subsequent neutralization of the mixture by Na2CO3 and re-filtration through 0·2 m filter to clarify the extract. Samples containing a considerable amount of sand were dry-sieved through a series of Wentworth sieves spaced at 1 phi intervals between 1 and 4 phi (Nikolaidou et al., 1983). The grain analysis gave the type of sediment and it is characterized as a coarse poorly-sorted sediment occurring on exposed shores where the wave energy is high. More specifically the negative value of Phi quartile skewness (SKq= 0·425) indicates that the finer particles are better sorted than the coarser ones, while the relatively high value of slope (QD=1·15) indicates that the sediment is poorly-sorted. A multiple regression analysis was applied (Gieskes et al., 1988; Barlow et al., 1995; Roy et al., 1996) in order to describe and predict the relationships among
the pigments in SW and IW. The following equation was applied: Chl a=intercept+1 (Pigment 1) +2 (Pigment 2)+ . . ..+n (Pigment n) Estimating the significant (P<0·05) beta coefficients (i) through the stepwise forward multiple regression we could determine which variable is the most impactful contributor collectively to the total Chl a biomass (dependent variable). These coefficients should be used only as a guide to the relative importance of the independent variables (various pigments) included in the equation and only in the range of values for which sample data actually exist (Hair et al., 1998). Bivariate correlations (Pearson’s coefficients) were also calculated among the pigment concentrations in the IW and SW separately for each pigment, on a monthly basis, in order to test if the pigments in the two media are related.
Algal pigments of an Eastern Mediterranean coastal area 419 6 4 mAU
mAU
4 2
2
Zea
BUF
6
(b) Chl a
Chl c
(a)
0
0
–2
–2 –4 15
(c)
30
20
25
25
10
(d)
15
20 15 10 5
15
20
25
15
20
25
HEX
10 mAU
mAU
5
0
Fuco
10
Chl b
5
Per
0
5 0
0 –5
–5 0
5
10
15
20
25
Time (min)
0
5
10
Time (min)
F 2. Selected chromatograms from sea water (a), interstitial water (b) and trays before immersion (c) and after immersion (d).
Results The grain size distribution of the sediment in seabed and trays are given in Table 1 and a summary of physicochemical parameters in the water column in Table 2. The grain size distribution of the seabed sediment (as the average of two stations) showed that the fraction of particles having diameter bigger than 0·125 mm was 98·9% while the same fraction was 99·4% in sediment filled the trays. Although the signature pigments are used as an index to assess or characterize the phytoplankton community (Gieskes & Kraay, 1983; Roy et al., 1996; Poister et al., 1999) there is not always a well documented relationship between the algal biomass and pigment concentrations through the different studies. The lack of specificity of some signature pigments and failings in methods like the coelutions e.g. Zea-luteinChl b or peak overlappings e.g. Chl a and pheo b can obscure this correlation. A first example in this investigation is the weak performance of peridinin detection in HPLC analysis which eluted in very low concentrations although a large population of dinoflagellates was found by microscopy (data in preparation). This discrepancy of peridinin detection may
be caused either by the presence of heterotrophic dinoflagellate species (Loret et al., 2000) or the existence of dinoflagellates that did not contain peridinin (Dodge, 1984). Examples of absorbance chromatograms are presented in Figure 2 showing the elution patterns of a range of chlorophylls and carotenoid pigments detected in November 1999 in SW (A) and IW (B), as well in trays (C) before immersion in May 1999, and (D) after immersion in July 1999. The elution patterns of pigments detected in trays before immersion (C) and after immersion (D) reveals the big difference in microbial and algal composition between them. On dry defaunated sand (C) the fucoxanthin and Chl a were detected in lower concentrations than Chl b while after immersion this wet sand (D) had a pigment composition very similar to that of seabed around the trays with only traces of Chl b. The identified pigments were chlorophylls (Chl a, Chl b and Chl c), zeaxanthin (Zea), peridinin (Per), fucoxanthin (Fuco), its derivatives 19 hexanoyloxyfucoxanthin (HEX) and 19 -butanoyloxyfucoxanthin (BUF), and pheophytins (Pheo a and Pheo b). We selected six pigments as chemotaxonomic markers of the major algal classes (pigment
420 A. Metaxatos & L. Ignatiades 10 000
Chl a
3
650
0
0
Chl b
0.2
1200
0.1
600
Chl c
2
600
1
300
0
0
Zea
0.4
0.2
–3
1300
0
1000
J FMAMJ J A S ON D
Per
0.3
7000
J FMAMJ J A S ON D
Fuco
0
0
5 3000
J FMAMJ J A S ON D
HEX
1.4
14 000
J FMAMJ J A S ON D
Pheo a
0
7
–3
SW pigments (mg m )
IW pigments (mg m )
5000
6
0.15 3500
500
0
J FMAMJ J A S ON D
0
0
2.5
J FMAMJ J A S ON D
0
0.7
1500
0
J FMAMJ J A S ON D
0
3.5
7000
0
J FMAMJ J A S ON D
0
F 3. The seasonal distribution of pigments in the three layers of the interstitial water/sediment system (IW: 0–3 mm, 10–13 mm, 30–33 mm) and sea water (SW: 0–8 m). 30–33 mm; 10–13 mm; 0–3 mm; — — SW.
signatures or biomarkers) and these included Chl a as an indicator of total phytoplankton biomass, Per (dinoflagellates), Fuco (diatoms), HEX (prymnesiophytes), and Zea (blue-green algae). However, the lack of specificity of some markers e.g. Fuco reduced the taxonomic precision from the pigment approach (Roy et al., 1996). It is worth noticing that the chromatogram [Figure 2(c)] of the trays before immersion (defaunated sand) showed a variety of pigments which might be produced from algae or bacteria that could survive on the dry sand. The seasonal distribution of pigments in the three interstitial water/sediment layers (IW: 0–3 mm, 10– 13 mm, 30–33 mm) and the sea water (SW: 0–8 m) are given in Figure 3. In SW all pigments showed a major peak in September and several minor peaks in various seasons without presenting a uniform seasonality. In September the pigment concentrations were very high (Chl a: 4·87 mg m 3; Fuco: 4·35 mg m 3). A minor peak of Chl a (0·67 mg m 3) coincided with a minor peak of HEX (0·26 mg m 3) in April. In July a minor Chl c peak was observed (0·12 mg m 3) and later in October two other peaks were recorded (Zea: 0·18 mg m 3, Pheo a: 0·74 mg m 3). Chl a, Pheo a and Fuco values were high throughout the year while BUF, Per and Chl b values were low. A marked seasonal variation of total pigment concentrations in IW (0–33 mm) was also recorded without a defined profile (Figure 3). Total maxima were found during the period April–May in the pigments: BUF (891 mg m 3 ), HEX (3035 mg m 3), Chl b
(1198 mg m 3), Pheo a (12586 mg m 3), during July–August in: Chl a (8378 mg m 3), Chl c (1052 mg m 3) Fuco (6202 mg m 3) and during November–December in Per (896 mg m 3) and Zea (519 mg m 3). The vertical distributions of various pigments in the three layers fluctuated on a monthly basis giving various patterns. Only in a few cases were certain pigments (Chl a, Chl c, Fuco and HEX) equally distributed in the three layers (Chl c: June–July, Fuco: January–December, HEX: March, Chl a: April–May– January) while the distribution between layers was unequal during the rest of the year. In a few exceptional cases, an unusually high accumulation was registered. For example in May, Per (peridinin) concentration reached up to 77% at the deeper layer whereas in summer it was accumulated (80%) at the top (Figure 3). Pigment annual concentrations, their ranges and the percentage relationship of the pigments among layers are given in Table 3. When the annual average percentages at the three layers were evaluated only HEX showed almost equal percentages (31%–34%– 35%) from the top to the deeper layer. On the other hand, some pigments presented a sharp decline from the top to the deeper layer (Pheo a: 51%–32%–17%, Zea: 40%–33%–27%, Chl b: 46%–37%–23%), or a minor decline (BUF: 38%–37%–25% , Chl a: 39%– 32%–30%). Chl c and Fuco exhibited an increase from the top layer to the deeper one (Chl c: 31%– 30%–38%, Fuco: 32%–30%–38%) whereas Per
1983 (951–3481) 39% 1606 (665–2520) 32% 1538 (670–2984) 30%
51271904 1709240 1025 238–1637 0·76 (0·25–4·87)
Intermediate layer (10–13 mm) Mean (mg m 3) Range Percentages
Deeper layer (30–33 mm) Mean (mg m 3) Range Percentages
IW (0–33 mm) Sum of three layers Mean (mg m 3)SD
Average of three layers Mean (mg m 3)SD
Trays Mean (mg m 3) Range
Sea water (0–8 m) Mean (mg m 3) SD Range
Chl a
Top layer (0–3 mm) Mean (mg m 3) Range Percentages
Pigments
0·16 (0·03–1·21)
212 24–356
20318
609332
223 (51–429) 38%
189 (36–334) 30%
196 (70–419) 31%
Chl c
0·05 (0–0·2)
37 0–91
12420
362344
107 (0–615) 23%
120 (0–312) 37%
146 (0–363) 46%
Chl b
0·08 (0·01–0·35)
129 40–200
10223
306151
81 (23–172) 27%
98 (45–236) 33%
126 (23–258) 40%
Zea
(0–0·02)
10 0–20
7238
197254
39 (0–206) 25%
62 (0–183) 37%
114 (0–788) 38%
BUF
0·17 (0·01–1·35)
384 45–648
424106
1228745
530 (60–1617) 35%
317 (135–651) 34%
425 (0–1063) 31%
HEX
0·03 (0–0·29)
270 53–480
11571
344312
121 (0–779) 34%
40 (0–203) 21%
183 (0–576) 45%
PER
0·82 (0–6·59)
402 0–714
1445571
43363273
897 (0–4124) 17%
1403 (241–3391) 32%
2031 (906–5072) 51%
Pheo a
0·49 (0·02–4·35)
1268 137–2320
1183144
35491362
1344 (837–1954) 38%
1066 (433–1687) 30%
1139 (338–2561) 32%
Fuco
T 3. Pigment annual means, ranges and percentages (%) in interstitial water/sediment system (IW) and annual means and ranges in sea water (SW) and trays. Data are averages from the two stations S1 and S2
Algal pigments of an Eastern Mediterranean coastal area 421
422 A. Metaxatos & L. Ignatiades T 4. Pigment ratios in sea water (SW) and interstitial water/sediment (IW) Pigment ratios Medium
Fuco/Chl a
Chl c/Chl a
HEX/Chl a
SW
IW
SW
IW
SW
IW
SW
IW
SW
IW
SW
IW
January February March April May June July August September October November December Mean
0·26 0·56 0·09 0·16 0·39 0·16 0·26 0·63 0·89 0·27 1·09 0·28 0·42
0·67 0·71 0·77 0·66 0·36 0·56 0·54 0·76 0·99 0·80 0·90 0·82 0·81
0·09 0·19 0·22 0·12 0·13 0·15 0·43 0·25 0·25 0·10 0·24 0·09 0·19
0·09 0·13 0·10 0·19 0·05 0·15 0·13 0·14 0·18 0·21 0·09 0·03 0·12
0·11 0·19 0·04 0·39 0·08 0·22 0·15 0·15 0·28 0·12 0·03 0·21 0·17
0·05 0·13 0·31 0·57 0·20 0·34 0·18 0·17 0·30 0·41 0·40 0·05 0·26
0·01 <0·01 <0·01 0·03 <0·01 <0·01 <0·01 <0·01 <0·01 0·01 0·02 <0·01 0·01
<0·01 <0·01 0·03 0·17 0·02 0·03 0·04 0·04 0·08 0·02 0·02 <0·01 0·04
0·16 0·08 0·05 <0·01 0·04 0·05 0·05 0·08 0·04 0·10 0·10 0·43 0·10
<0·01 0·01 0·05 0·15 0·17 0·11 0·07 0·03 0·03 0·08 0·09 0·04 0·07
0·28 0·05 <0·01 0·71 0·19 1·17 0·81 1·25 1·35 2·17 0·35 1·47 0·82
0·36 0·36 1·07 1·59 1·82 0·80 0·27 0·30 0·61 1·60 0·85 1·04 0·89
showed a different pattern since its annual mean percentage was higher at the top (45%), that at the deeper layer was next (34%) and that in the intermediate layer was lower (21%). The pigment/Chl a concentration ratios in the sea water (SW) and the interstitial water/sediment layers (0–33 mm) from all sampling periods are shown in Table 4. The ratio is a convenient index of the relative concentration of both pigments, reducing the variance between consecutive chromatographic analyses when different samples are compared (Vernet & Lorenzen, 1987). These ratios displayed a variability throughout the year in both media, IW and SW, without a distinct profile. In SW ratios ranged from <0·01 to 2·17 and in IW from <0·01 to 1·82. High ratios were encountered in SW and IW during autumn (SW: Fuco/Chl a: 1·09; Pheo a/Chl a: 2·17 and IW: Fuco/Chl a: 0·99; Chl c/ Chl a: 0·21) and certain ratios exhibited their maxima during spring (HEX/Chl a=0·57, BUF/Chl a=0·17, Pheo a/Chl a=1·82 and Chl b/Chl a=0·17) only in IW. Figure 4 focuses on the seasonality of selected pigments (Chl a, Chl c, HEX, Zea, Pheo a, Fuco) in the interface of the two media e.g. the near bottom water (NBW) and the top layer (0–3 mm) of interstitial water/sediment system by using the mean values of the two stations S1 and S2. Fuco showed a peak in the top layer of IW in August (2560 mg m 3) followed by a NBW peak in September (4·1 mg m 3) and a similar pattern followed by Chl c and Chl a. Pheo a gave two different peaks in May in the top layer (5072 mg m 3) and a second in September in NBW (7·2 mg m 3) without any time coincidence. According to Figures 3 and 4 two general features were identified: the existence of a time lag between the formation of pigment peaks (with the IW peak
Buf/Chl a
Chl b/Chl a
Phaeo a/Chl a
being always first), and a difference in the seasonal distribution of peaks among pigments. The multiple regression analysis could estimate and define the quantitative relationships by revealing statistically significant differences in pigment contributions to Chl a biomass among various layers of IW or depths in SW and between media which might not be defined by ratios or correlations. According to this analysis (Table 5) Fuco exhibited a strong contribution with Chl a biomass in all layers of IW. In the top layer (0–3 mm) HEX (a marker for the water column phytoplankton) had a negative relationship with Chl a. In the intermediate layer (10–13 mm) Chl b had a positive relationship while Zea had a negative relationship to Chl a biomass. It is worth noticing that in the deeper layer (30–33 mm) a positive relationship of BUF and Pheo a with Chl a were found. This analysis also gave a positive relationship of Chl a with Chl c in SW and with HEX in NBW. On the other hand, the Pearson’s correlation analysis showed no significant correlation among pigments in the SW and IW. Discussion The present investigation focused on the estimation of various critical parameters in the interstitial water (IW) and water column (SW) systems affecting the pigment seasonal distribution in both media. The values of physicochemical parameters (Tables 1 and 2) were similar at the two stations S1, and S2, and according to the nutrient levels in SW (N-NO3: 0·07– 6·1 g-at; P-PO4: 0·04–4·1 g-at) the area could be characterized as inshore eutrophic (Ignatiades et al., 1992). Pigments in the IW can be derived from
Algal pigments of an Eastern Mediterranean coastal area 423 4.5
4500
1
450
Chl a
Chl c 3
3000
300 0.5
1.5
–3
0
150
0
1200
1.2
0
0
300
HEX
0.4 Zea
800
200 0.6
400
0.2 100
0
3000
4.5
0
0
6000
Fuco
–3
0
Pigments of NBW (mg m )
Pigments of IW (TL) top layer (mg m )
1500
8 Pheo a
2000
3
4000 4
1000
1.5
0
0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
0
2000
F 4. Seasonal variation of pigments in the top layer of IW (0–3 mm) and near bottom water (NBW).
NBW;
TL.
T 5. Parameters of the multiple regression equations for the SW (0–8 m), NBW, IW (0–33 mm) and the three layers of IW. Only the significant (P<0·05) beta coefficients () have been given Beta coefficients Source SW (0–8 m) NBW IW (0–33 mm) IW:TL (0–3 mm) IW:IL (10–13 mm) IW:DL (30–33 mm)
Fuco
HEX
Chl c
Chl b
Zea
BUF
Pheo a
Intercept
R2
P
0·694
0·059 0·278 136·100 455·100 685·300 65·000
0·988 0·988 0·710 0·960 0·950 0·820
0·000 0·000 0·000 0·001 0·016 0·010
0·975 0·668 0·822 0·836 0·478
0·861 0·330 0·710 0·739
microphytobenthos grown in situ, macroalgae detritus and sedimented phytoplankton and tychoplankton (Lucas & Holligan, 1999). The sandy bottoms of the two stations S1 and S2 had a similar grain size distribution with the fraction of grains (>0·5 m) being about 57% (Table 1). The trays had a coarser deposit with this fraction being greater than 75·5%. Although the defaunated sand
0·490 0·701
filling the trays before immersion was collected from the seabed of the sampling stations, a different grain sorting was produced gradually by the water movement in their restricted volume in relation to seabed (Table 1). Thus, the different levels of pigments (Table 3) recorded in the trays can be related to their different grain size distribution. Snelgrove and Butman (1994) suggest that grain size covaries with
424 A. Metaxatos & L. Ignatiades
porewater chemistry and microbial abundance and composition which could directly or indirectly influence infaunal populations. In the literature there is a contradiction regarding the relationship between the type of sediment and its composition of algae. Lucas and Holligan (1999) found greater Chl a in siltier sites than sandier ones and they also recorded a positive correlation between Chl a and silt. Furthermore, these authors reported that on the Molenplaat tidal flat (SW Netherlands) Fuco/Chl a ratios (0·35–1·60) indicated the dominance of diatoms and that the distribution of Chl a, Fuco and other pigments were correlated with grain size of sediments. However, in Port Phillip bay (S. Australia) no significant differences were found in Pheo a and Chl a values and ratio of total organic matter to Chl a across various sediment types (Light & Beardall, 1998). One explanation for these inconsistencies is that grain size is not an adequate descriptor of the sedimentary environment. Thus, the water flow on the tray surface (the trays protruded about 10 cm from the surrounding bottom) might be different from the encircling seabed altering the interfacial transport of algal cells into sediment. In addition to their taxonomic role, pigments can provide information on the physiological condition of algae (Hallegraeff & Jeffrey, 1984) and on the fate of these cells (Barlow et. al., 1995). More specifically, Chl a is commonly used as an indicator of phytoplankton biomass while the concentrations of its degradation products (for example Pheo a) are used as diagnostic indicators of physiological status, detrital content and grazing processes in natural phytoplankton communities (Mantoura & Llewellyn, 1983). There is no previous mention in the literature for pigment seasonality in the IW and the available information on the pigment distribution in sediment on a monthly basis is scarce (Abele, 1991; Sun et al., 1994). In the present investigation the temporal variations of IW pigments did not follow their seasonal variation in SW since in SW (0–8 m) and NBW (Figures 3 and 4) all pigments showed a peak in September but in IW the pigments displayed a various seasonality without a defined profile, resulting in the lack of any significant correlation between the two media. Also, there seemed to be a time lag between maximum values in SW and IW with the peaks in IW preceding those in SW (Figure 3). A similar conclusion has been reported by Poister et al. (1999) who found that the period of increased chlorophyll concentration in the water column corresponded to a period of decreased chlorophyll sedimentation rate and changes in the rates of pigment sedimentation did not reflect changes in pigment concentration in the water column of Trout Lake (Wisconsin, USA). It was
also reported (Rodriguez & Guerrero, 1994) that in Malaga Bay no clear temporal correlation between the water and the sediment pigment concentrations could be seen, even when the time-lag of water/sediment interface processes was considered. Special emphasis was given in the analysis of data derived from the top layer of IW and NBW e.g. the interface of two media. The pigment variation in NBW and top layer (TL, IW) gave a variety of patterns (Figure 4). NBW however, was not only affected by fluxes from beneath through TL but also from settling material from the water column which was mixed with resuspended particulate matter. In shallow waters wave action and currents will often be the dominating factors for particle fluxes across the interface (Graf & Rosenberg, 1997). Factors such as lateral redistribution of particles and different degradation rates between pigments may alter any simple correlation of water column and IW composition (no significant correlation between pigments found in our analysis) and therefore any similarity of pigment seasonality. This sound difference in temporal and spatial pigment fluctuation and magnitude between the two systems SW and IW can be additionally related to benthic in and epifauna activities. Gravitational settling, filtration activities of benthic animals and aggregation at the sediment–water interface withdraw particles from the NBW (Huettel et al., 1996). Fuco was the most abundant accessory pigment in all media giving a total average 0·49 mg m 3 in SW, 1183 mg m 3 in IW and 1268 mg m 3 in trays (Table 3, Figures 3 and 4). This can be due to an accumulation of benthic and pelagic diatoms on this sandy site, although their origin as benthic or pelagic can not be identified precisely. Our results support the aspect that coarse sandy sediment in shallow waters provides ideal substrate for benthic diatoms rich in Fuco (Abele, 1991) and that diatoms generally contribute a greater proportion of their biomass to sediments relative to other phytoplankton (Reynolds et al., 1982). Furthermore, Fuco was also found as the dominant carotenoid pigment of deposits in various depths of Cretan Sea (Duineveld et al., 2000) as well as in silty and sandy sediments in Westerschelde estuary (Lucas & Holligan, 1999). According to Bianchi et al. (1993) the benthic diatoms are considered as high quality (low C/N ratio) living resources and one of the most nutritious foods for marine and aquatic invertebrates. Pheo a was found in most samples and it was an abundant pigment in both media reaching very high values (6·59 mg m 3 in SW and 5072 mg m 3 in IW). The levels of this pigment tended to elevate in IW in spring when Chl a decreased and benthic macrofaunal activity was high.
Algal pigments of an Eastern Mediterranean coastal area 425
Light and Beardall (1998) found in Port Phillip bay that significantly greater Pheo a was associated with shallow sites and the uppermost sediment stratum than in deep water sites and lower strata. As far as vertical distribution of pigments in IW layers is concerned it was shown (Table 3) that Chl a (39% TL, 32% IL, 30% DL) profiles exhibited a slight decrease with depth from TL (0–3 mm) to DL (30–33 mm) contrasting the results of MacIntyre and Cullen (1995) and Lucas and Holligan (1999) who also examined the vertical profiles in the upper (0–3 cm) sediment layers and found a rapid decrease of Chl a over the top 1 cm. Also, Sun et al. (1994) found a nearly exponential decrease of Chl a within the upper 4 cm in Long Island. The profiles of Chl b (46% TL, 37% IL, 23% DL), BUF (38% TL, 37% IL, 25% DL) and Pheo a (51% TL, 32% IL, 17% DL) showed a sharper decline but Fuco (32% TL, 30% IL, 38% DL) and Chl c (31% TL, 30% IL, 38% DL) exhibited a slight increase with depth. The penetration distribution changes on a monthly basis (Figure 3) exhibited some extreme accumulations in the top (Chl c: May; Per: June, July, August, November) or in the deeper layer (Pheo a: January, February; Per: May; Chl c: June, July). According to Lucas and Holligan (1999) the deposition of pigments in the sediment is a complex phenomenon. For example, the sinking-sedimented phytoplankton such as dinoflagellates (Per) or prymnesiophytes (HEX) tended to be accumulated in the top layer (Figure 3) and the observed from time to time high levels of peridinin in the intermediate and deeper layer might be due to various reasons e.g. the vertical migration (phototaxis), permeability of the sediments and bottom currents, but there no available data for these parameters. On the other hand, Fuco, Chl a and Chl b are short lived markers associated with recent phytoplankton sedimentation events (Abele, 1991) decreasing sharply with depth. Factors like sediment type, depth, resuspension, grazing, as well as degradation rates, transformations in situ and half-life of pigments seem to be critical for the temporal and spatial distribution of pigments in the sediments. Interpretation of pigment ratios (Table 4) must be carried out with a degree of caution because this approach lacks statistical validation. Nevertheless, we estimated the ratios of six pigments and we draw some useful conclusions e.g. the phaeo a/Chl a ratios provide an indication of the physiological state of benthic microalgal population, where high values correspond to stressed or declining populations as mentioned by Light and Beardall (1998). In the present study, the major pigment peaks were recorded in SW in September and they were followed by a very high ratio
Pheo a/Chl a (SW: 2·17; IW: 1·6) in both media that could be associated with the declining populations in October. Also, the high Fuco/Chl a ratios in both media (SW: 0·42, IW: 0·81) of our area might reflect the dominance of diatoms whereas the low BUF/Chl a and Chl b/Chl a ratios might show the shortage of the taxa having the relevant pigments. The pigment analysis data of this work demonstrate that the algal communities thriving in the strongly interacting sea water and interstitial water/sediment system have a differing seasonally quantitative and qualitative composition. This might be attributed to the action of the sand bed as a gigantic filter that accumulates the algal cells forming thus a distinct community structure.
References Abele, D. 1991 Potential of some carotenoids in two recent sediments of Kiel Bight as biogenic indicators of phytodetritus. Marine Ecology Progress Series 70, 83–92. APHA, AWWA, WEF 1992 Standard Methods for the Examination of Water and Wastewater, 18th edn. American Public Health Association, Publication Office, Washington DC, pp. 2:53–2:58. Barlow, R. G., Mantoura, R. F. G., Peinert, R. D., Miller, A. E. J. & Fileman, T. W. 1995 Distribution, sedimentation and fate of pigment biomarkers following thermal stratification in the western Alboran Sea. Marine Ecology Progress Series 125, 279–291. Bayne, B. L., Hawkins, A. J. S. & Navaro, E. 1987 Feeding and digestion by the mussel M. edulis (Bivalvia:Mollusca) in mixtures of silt and algal cells at low concentrations. Journal of Experimental Marine Biology and Ecology 111, 1–22. Bianchi, S. T., 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. Bianchi, S. T., Demetropoulos, A., Hadjichristophorou, M., Argyrou, M., Baskaran, M. & Lambert, C. 1996 Plant pigments as biomarkers of organic matter sources in sediments and coastal waters of Cyprus (Eastern mediterranean). Estuarine, Coastal and Shelf Science 42, 103–115. Bodoy, A. & Plante-Cuny, M. R. 1984 Relations entre l’evolution saisonniere des populations de palourdes (Ruditapes decussatus) et celle des microphytes benthiques et planctoniques (Golfe de Fos, France). Haliotis 14, 71–78. Botto, F. & Iribarne, O. 2000 Contrasting effects of two burrowing crabs (C. granulata and U. uruguayensis) on sediment composition and transport in estuarine environments. Estuarine, Coastal and Shelf Science 51, 141–151. Buffan-Dubau, E. & Carman, K. R. 2000 Extraction of benthic microalgal pigments for HPLC analyses. Marine Ecology Progress Series 204, 293–297. Dodge, J. D. 1984 Dinoflagellate taxonomy. In Dinoflagellates (Spector, D. L., ed.). Academic Press, London, pp. 17–38. Duineveld, G. C. A., Tselepides, A., Witbaard, R., Bak, R. P. M., Berguis, E. M., Nieuwland, G., van der Weele, J. & Kok, A. 2000 Benthic-pelagic coupling in the oligotrophic Cretan Sea. Progress in Oceanography 46, 457–480. Gieskes, W. W. C. & Kraay, G. W. 1983 Unknown chlorophyll a derivatives in the North Sea and the tropical Atlantic Ocean revealed by HPLC analysis. Limnology and Oceanography 28, 757–766.
426 A. Metaxatos & L. Ignatiades Gieskes, W. W. C., Kraay, G. W., Nontji, A., Setiapermana, D. & Sutomo, 1988 Monsoonal alternation of a mixed and a layered structure in the phytoplankton of the euphotic zone of the Banda Sea (Indonesia): a mathematical analysis of algal pigment fingerprints. Netherlands Journal of Sea Research 22, 123–137. Graf, G. & Rosenberg, R. 1997 Bioresuspension and biodeposition: a review. Journal of Marine Systems II, 269–278. Hair, F. J., Anderson, E. R., Tatham, L. R. & Black, C. W. 1998 Multivariate Data Analysis, fifth edition. Prentice-Hall International Inc., pp. 730. Hallegraeff, G. M. & Jeffrey, S. W. 1984 Tropical phytoplankton species and pigments of continental shelf waters of N. and NW Australia. Marine Ecology Progress Series 20, 59–74. Hawkins, A. J. S. & Bayne, B. L. 1992 Physiological interrelations and the regulation of production. In The Mussel Mytilus: Ecology, Physiology, Genetics and Culture (Gosling, E., ed.). Elsevier, Amsterdam, pp. 171–212. Huettel, M. & Rusch, A. 2000 Transport and degradation of phytoplankton in permeable sediment. Limnology and Oceanography 45 (3), 534–549. Huettel, M., Ziebis, W. & Forster, S. 1996 Flow-induced uptake of particulate matter in permeable sediments. Limnology and Oceanography 41 (2), 309–322. Ignatiades, L., Karydis, M. & Vounatsou, P. 1992 A possible method for evaluating Oligotrophy and Eutrophication Based on Nutrient Concentration scales. Marine Pollution Bulletin 24 (5), 238–243. Jørgensen, C. B. 1990 Bivalve Filter Feeding, Hydrodynamics, Bioenergetics, Physiology and Ecology. Olsen & Olsen, Fredensborg, Denmark. Levinton, J. S. & McCartney, M. 1991 Use of photosynthetic pigments in sediments as a tracer for sources and fates of macrophyte organic matter. Marine Ecology Progress Series 78, 87–96. Liddicoat, M. I., Tibbits, J. S. & Buttler, I. E. 1976 The determination of ammonia in natural waters. Water Research 10, 567–568. Light, B. & Beardall, J. 1998 Distribution and spatial variation of benthic microalgal biomass in a temperate, shallow-water marine system. Aquatic Botany 61, 39–54. Loret, P., Pastoureaud, A., Bacher, C. & Delesalle, B. 2000 Phytoplankton composition and selective feeding of the pearl oyster Pinctada margaritifera in Takapoto lagoon (Tuamotu Archipelago, French Polynesia): in situ study using optical microscopy and HPLC pigment analysis. Marine Ecology Progress Series 199, 55–67. Lucas, C. H. & Holligan, P. M. 1999 Nature and ecological implications of algal pigment diversity on the Molenplaat tidal flat (Westerschelde estuary, SW Netherlands). Marine Ecology Progress Series 180, 51–64. MacIntyre, H. L. & Cullen, J. J. 1995 Fine scale vertical resolution of chlorophyll and photosynthetic parameters in shallow-water benthos. Marine Ecology Progress Series 122, 227–237. Mantoura, R. F. C. & Llewellyn, C. A. 1983 The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by HPLC. Analytica Chimica Acta 151, 297–314. Moore, B. H. 1962 Marine Ecology. John Wiley & Sons, NY, 493 pp.
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Seasonality of Algal Pigments in the Sea Water and Interstitial Water/Sediment System of an Eastern Mediterranean Coastal Area A. Metaxatos and L. Ignatiades NCSR ‘ Demokritos ’, Institute of Biology, Aghia Paraskevi, 15310 Attiki, Greece Received 20 February 2001 and accepted in revised form 1 October 2001 An effort has been made herein to describe the seasonal variation of the microalgal pigments, nutrient concentrations and other physicochemical parameters in a sandy and eutrophic coastal area of North Euboikos gulf, Aegean Sea. The algal pigments chlorophyll a, chlorophyll b, chlorophyll c, fucoxanthin, zeaxanthin, peridinin, 19 -hexanoyloxyfucoxanthin, 19 -butanoyloxyfucoxanthin, pheophytin a and pheophytin b were analysed by High Performance Liquid Chromatography (HPLC) in the interstitial water/sediment system (IW) and sea water (SW) during 1999. Chl a was recorded in all samples and its annual mean values were 1709 mg m 3 in IW and 0·76 mg m 3 in SW. Fucoxanthin was the most abundant accessory pigment in both media giving a total average 1183 mg m 3 in IW and 0·49 mg m 3 in SW while the pheophytin a mean values were 1183 mg m 3 in IW and 0·49 mg m 3 in SW. The vertical distributions of various pigments in the three layers of IW (0–3 mm, 10–13 mm and 30–33 mm) fluctuated quantitatively on a monthly basis. Only in a few cases certain pigments (Chl a, Chl c, Fuco and HEX) were equally distributed in the three layers (Chl c: June–July, Fuco: January–December, HEX: March, Chl a: April–May–January), while the distribution between layers was unequal during the rest of the year. Special emphasis was given in the analysis of data derived from the top layer (0–3 mm) of interstitial water/sediment system (IW) and near bottom water (NBW) e.g. the interface of two media. The ratios of pigment/Chl a and correlations were also estimated in order to enlighten the relationships among various pigments spatially and temporally. By using stepwise forward multiple regression we determined the pigments (Fuco, HEX, Chl c and BUF) that are the significant contributors collectively to the total Chl a biomass and we revealed differences in pigment contributions among various layers of IW or depths in SW and between media. 2002 Elsevier Science Ltd. All rights reserved.
Keywords: algal pigments; HPLC; interstitial water/sediment system; Euboikos gulf; Aegean Sea
Introduction The sea bottom environment of inshore waters and its relationship with the water column above it has been the subject of numerous studies. These entities are not completely independent, since the water contributes by exchange to the interstitial water (water content of the sediment) and receives material from it (Moore, 1962). In permeable beds advective interfacial water can flow through them providing a fast carrier for the exchange of substances between the water column and the upper sediment layers, so that permeable shelf sands represent gigantic filter systems (Huettel & Rusch, 2000). Thus, in shelf environments, up to 50% of the primary production can settle through the water column and most of this particulate organic material is mineralized in the sediment, and its decomposition products are returned to the water column (Huettel et al., 1996). Benthic microalgal communities include both motile algae (epipelic diatoms, cyanobacteria, 0272–7714/02/090415+12 $35.00/0
flagellates, etc.) as well as sessile algae attached to the sand grains (epipsammic diatoms, sporelings) which build up an important autotrophic biomass for the ecosystem, with values of up to 560 mg m 2 in the upper centimetre of sediments (Wolfstein et al., 2000). These communities are also involved in many fundamental processes like the production of labile matter, the regeneration of nutrients and the regulation of oxygen and they constitute an important source of food for heterotrophic mollusca like suspension-feeding bivalves (Bodoy & Plante-Cuny, 1984; Bayne et al., 1987; Jørgensen, 1990; Hawkins & Bayne, 1992). Their contribution to the carbon budgets of shallow-water systems has been extensively documented (Varela & Penas, 1985). Due to the relevance of benthic primary producers to the ecosystem, estimating their abundance in sea water as well as in the upper centimetre of sediment and its interstitial water altogether (or the interstitial water/sediment system) remains an important goal. In the literature, information on the pigment content of 2002 Elsevier Science Ltd. All rights reserved.
416 A. Metaxatos & L. Ignatiades N
39 Eu
boi
38 ge
37
an Se
Euripus Canal
a
s S1 S2
Ae
ko
36 35
21 22 23 24 25 26 27 28
Saronicos Gulf
Aegean Sea
F 1. Map of the study area in North Euboikos gulf, Aegean Sea.
sediments are mostly referred to Chl a determinations or its degradation products (Sun et al., 1991, 1994; MacIntyre & Cullen, 1995; Duineveld et al., 2000) whereas studies on other major or accessory pigments are scanty (Abele, 1991; Levinton & McCartney, 1991; Lucas & Holligan, 1999). Also, compared to the great number of results published worldwide, information on pigment content of the sediments in the Mediterranean Sea is poor and limited to the western areas (Rodriguez & Guerrero, 1994; Barlow et al., 1995) whereas information from the eastern coastlines of this oligotrophic sea is scant (Bianchi et al., 1996; Duineveld et al., 2000). The main objective of this study was to present a complete description of the dominant and accessory pigments (chlorophyll a, chlorophyll b and chlorophyll c, fucoxanthin, zeaxanthin, peridinin, 19 -hexanoyloxyfucoxanthin, 19 -butanoyloxyfucoxanthin, pheophytin a and pheophytin b) in sea water and in the interstitial water/sediment system of Euboikos gulf, Aegean Sea (Eastern Mediterranean). This inshore environment, although it is occupied by large stocks of bivalves, is unexplored regarding the important parameters such as phytoplankton biomass, grain size distribution and the related physicochemical conditions that might affect infaunal populations. Material and methods Study site North Euboikos gulf (Aegean Sea) extends along the western coast of Evia island, which is separated from
the Greek mainland by the Euripus channel that is 40 m wide, 60 m long and 8 m deep. Two inshore stations 350 m apart were selected: S1 having maximum depth of 3·5 m and S2 having maximum depth of 8 m (Figure 1). The benthic community in this coastal area is dominated by bivalves (Callista chione, Venus verrucosa, Loripes lacteus, Loripes fragilis, Hiatella arctica, Dosinia exoleta, Acanthocardia sp., Parvicardium sp. etc.). The stations represent the limits of C. chione’s geographical distribution, which is the most abundant bivalve regarding the biomass (dry weight: 154·3 gr m2) with an average population density of 6 individuals m2 (data in preparation).
Water sampling and analysis Sea water samples (SW) were collected monthly during 1999 with a Hydro-bios sampler equipped with an inverted thermometer from two depths (a) surface sea water (SSW: 0·3 m below the surface) and (b) near bottom water (NBW: 0·3 m above the seabed). Vertical profiles of temperature (inverted thermometer), salinity and dissolved oxygen were measured in situ with portable apparatus (LaMotte, Portable Electronic Lab., model DCL-05). Samples for the nutrient analyses were stored in 150 ml polyethylene bottles and kept frozen at 30 C until analysis of phosphates, nitrates, nitrites, silicates (Strickland & Parsons, 1974) and ammonia (Liddicoat et al., 1976). Samples for phytopigment analysis were kept at 4 C in the dark and filtered through millipore
Algal pigments of an Eastern Mediterranean coastal area 417
filters (0·2 m pore size). The filters were stored in aluminium foil and kept at 90 C until further analysis by reverse-phase HPLC (model HP 1100 series) with a column Spherisorb ODS2 25 cm4·6 mm ID, 5 m particle size with a loop 50 l. Pigment detection was at 440 nm and the solvent system, flow rate and gradient system was according to Wright et al. (1991). Pigments were extracted from thawed filters using 0·5 ml 90% acetone at 4 C in the dark for several hours. For ease of comparison pigment concentrations are reported in mg m 3 in both media e.g. mg of pigments in one cubic metre of SW or mg of pigments in one cubic metre of IW (grains and interstitial water). Samples for estimation of total suspended solids (TSS) and ash free dry weight (AFDW) in sea water were kept at 4 C in the dark. A well-mixed subsample was filtered through a weighed standard glass-fibre filter and the residue retained on the filter was dried to a constant weight at 75 C. The increase in weight of the filter represented the total suspended solids (APHA et al., 1992). Afterwards, the residue was ignited to constant weight at 50050 C and the remaining solids represented the ash-weight while the weight lost on ignition represented the ash-free dry weight which is a rough approximation of the amount of organic solids (APHA et al., 1992; Botto & Iribarne, 2000). Sediment sampling Sediment for interstitial water/sediment (IW) analyses was collected by divers in 3–5 acrylic syringes (32 mm inner diameter) that were pushed into the sandy bottom, sealed in situ (for retaining both sediment and its interstitial water) and frozen in liquid nitrogen on board. To avoid mixing of the layers during transportation to the laboratory the samples were kept perpendicular. The cores were sectioned in 3 mm slices with a methanol-rinsed razor blade and care was taken to ensure that the sediment surface was perpendicular to the long axis of the cores to minimize unevenness in the thickness of the surficial layer (MacIntyre & Cullen, 1995). The slices (0–3 mm, 10–13 mm and 30–33 mm) were kept at 90 C until further analysis. A series of experiments was also performed in trays used in experimental bivalve aquaculture in order to evaluate the entire population of algae derived from the water column or resuspension from the seabed. Thus, in May 1999 a total of 35 plastic trays (30 cm20 cm13 cm) were filled with defaunated sand and secured on the surface of the seabed of the two stations S1 and S2 by two metal pegs anchoring in
the deep sediment. To prevent sediment loss during diving operations the trays were sealed in plastic bags which were later removed by divers on the sea bottom. The first tray sampling was in June 1999 and subsequent samplings were carried out monthly for 6 months thereafter. No sampling was carried out in December 1999 due to a storm after which all experimental trays were lost. Defaunated sand was obtained by air drying the sediment under shade for 1 month. Before use, this air-dried sand was examined under a microscope to ensure that there were no live macroinfauna, and it was also analysed for pigments. HPLC pigment analysis and sediment analysis HPLC pigment analysis was used to study the quantitative and qualitative pigment identification in the SW (sea water) and IW (interstitial water/ sediment system). The followed protocol (Wright et al., 1991) combined advantages of earlier methods and it was recommended by the SCOR Working group 78 (Wright & Jeffrey, 1997). This standard protocol makes use of the retention capacity of ammonium acetate-containing mobile phase and the special selectivity of acetonitrile-based eluents for carotenoids separation (Zapata et al., 2000). We used acetone instead of methanol as extraction solvent because it yielded significantly better recoveries of pheopigments while methanol as an extraction solvent may underestimate pheopigment concentrations as well as Chl a concentrations in benthic field samples (Buffan-Dubau & Carman, 2000). The slices of frozen interstitial water/sediment were added in equal volume to 90% acetone (sediment: acetone 90%, v/v, 1/1) for a direct extraction of pigments from the interstitial water contained in the sediment pores and most likely pigments attached to the surface of grains. This mixture was stirred and vortexed several times before the liquid phase was recovered from IW by centrifugation (5 min, 720g). The supernatant was kept at +4 C in the dark for several hours to complete pigment extraction and then it was centrifuged again to clarify the extract under the same conditions (ultrasonic bath was omitted to avoid pigment degradation). The final supernatant was injected into the HPLC system and detected at 440 nm (Wright et al., 1991). The extraction protocol presented here is similar to MacIntyre and Cullen (1995) and Light and Beardall (1998). Calibration was done with external standards obtained by the Sigma Chem. Co, and the International Agency for 14C determination, Denmark, except for pheophytin a and pheophytin b.
418 A. Metaxatos & L. Ignatiades T 1. The percentage distribution of grain diameters (Phi) in both stations (S1, S2) and trays Diameter of grain (mm)
Phi
S1 % meanSD
S2 % meanSD
Trays % meanSD
>2 >1 >0·5 >0·25 >0·125 >0·063 <0·063
1 0 1 2 3 4 >4
14·766·98 20·287·96 26·823·83 27·109·07 9·915·12 0·760·36 0·210·12
13·808·67 12·876·18 24·946·49 36·6213·72 10·695·86 0·830·43 0·260·24
19·347·50 31·201·97 25·914·06 17·514·03 5·451·17 0·390·09 0·140·04
T 2. The annual mean values and ranges of nutrients, temperature, salinity. Total suspended solids (TSS) and ash free dry weight (AFDW) in the two stations Nutrients (g-at l 1) Stations
N-NO2
N-NO3
N-NH4
P-PO4
SiO2
S1 S2 Ranges
0·110·10 0·110·10 0·02–0·402
1·321·30 1·321·38 0·07–6·1
1·351·68 1·331·77 0·22–6·19
0·390·90 0·360·96 0·04–4·1
4·691·92 4·841·90 1·46–7·96
Stations
T C
Salinity PSU
TSS (mg l 1)
AFDW (mg l 1
S1 S2 Ranges
20·506·6 20·506·5 11·1–29·6
37·500·70 37·500·90 36·8–38·2
0·0260·014 0·0230·009 0·0150·026
0·0180·12 0·0190·012 0·0130·021
These pigments were prepared in the laboratory by acidification (1 M HCl) of the corresponding chlorophylls and subsequent neutralization of the mixture by Na2CO3 and re-filtration through 0·2 m filter to clarify the extract. Samples containing a considerable amount of sand were dry-sieved through a series of Wentworth sieves spaced at 1 phi intervals between 1 and 4 phi (Nikolaidou et al., 1983). The grain analysis gave the type of sediment and it is characterized as a coarse poorly-sorted sediment occurring on exposed shores where the wave energy is high. More specifically the negative value of Phi quartile skewness (SKq= 0·425) indicates that the finer particles are better sorted than the coarser ones, while the relatively high value of slope (QD=1·15) indicates that the sediment is poorly-sorted. A multiple regression analysis was applied (Gieskes et al., 1988; Barlow et al., 1995; Roy et al., 1996) in order to describe and predict the relationships among
the pigments in SW and IW. The following equation was applied: Chl a=intercept+1 (Pigment 1) +2 (Pigment 2)+ . . ..+n (Pigment n) Estimating the significant (P<0·05) beta coefficients (i) through the stepwise forward multiple regression we could determine which variable is the most impactful contributor collectively to the total Chl a biomass (dependent variable). These coefficients should be used only as a guide to the relative importance of the independent variables (various pigments) included in the equation and only in the range of values for which sample data actually exist (Hair et al., 1998). Bivariate correlations (Pearson’s coefficients) were also calculated among the pigment concentrations in the IW and SW separately for each pigment, on a monthly basis, in order to test if the pigments in the two media are related.
Algal pigments of an Eastern Mediterranean coastal area 419 6 4 mAU
mAU
4 2
2
Zea
BUF
6
(b) Chl a
Chl c
(a)
0
0
–2
–2 –4 15
(c)
30
20
25
25
10
(d)
15
20 15 10 5
15
20
25
15
20
25
HEX
10 mAU
mAU
5
0
Fuco
10
Chl b
5
Per
0
5 0
0 –5
–5 0
5
10
15
20
25
Time (min)
0
5
10
Time (min)
F 2. Selected chromatograms from sea water (a), interstitial water (b) and trays before immersion (c) and after immersion (d).
Results The grain size distribution of the sediment in seabed and trays are given in Table 1 and a summary of physicochemical parameters in the water column in Table 2. The grain size distribution of the seabed sediment (as the average of two stations) showed that the fraction of particles having diameter bigger than 0·125 mm was 98·9% while the same fraction was 99·4% in sediment filled the trays. Although the signature pigments are used as an index to assess or characterize the phytoplankton community (Gieskes & Kraay, 1983; Roy et al., 1996; Poister et al., 1999) there is not always a well documented relationship between the algal biomass and pigment concentrations through the different studies. The lack of specificity of some signature pigments and failings in methods like the coelutions e.g. Zea-luteinChl b or peak overlappings e.g. Chl a and pheo b can obscure this correlation. A first example in this investigation is the weak performance of peridinin detection in HPLC analysis which eluted in very low concentrations although a large population of dinoflagellates was found by microscopy (data in preparation). This discrepancy of peridinin detection may
be caused either by the presence of heterotrophic dinoflagellate species (Loret et al., 2000) or the existence of dinoflagellates that did not contain peridinin (Dodge, 1984). Examples of absorbance chromatograms are presented in Figure 2 showing the elution patterns of a range of chlorophylls and carotenoid pigments detected in November 1999 in SW (A) and IW (B), as well in trays (C) before immersion in May 1999, and (D) after immersion in July 1999. The elution patterns of pigments detected in trays before immersion (C) and after immersion (D) reveals the big difference in microbial and algal composition between them. On dry defaunated sand (C) the fucoxanthin and Chl a were detected in lower concentrations than Chl b while after immersion this wet sand (D) had a pigment composition very similar to that of seabed around the trays with only traces of Chl b. The identified pigments were chlorophylls (Chl a, Chl b and Chl c), zeaxanthin (Zea), peridinin (Per), fucoxanthin (Fuco), its derivatives 19 hexanoyloxyfucoxanthin (HEX) and 19 -butanoyloxyfucoxanthin (BUF), and pheophytins (Pheo a and Pheo b). We selected six pigments as chemotaxonomic markers of the major algal classes (pigment
420 A. Metaxatos & L. Ignatiades 10 000
Chl a
3
650
0
0
Chl b
0.2
1200
0.1
600
Chl c
2
600
1
300
0
0
Zea
0.4
0.2
–3
1300
0
1000
J FMAMJ J A S ON D
Per
0.3
7000
J FMAMJ J A S ON D
Fuco
0
0
5 3000
J FMAMJ J A S ON D
HEX
1.4
14 000
J FMAMJ J A S ON D
Pheo a
0
7
–3
SW pigments (mg m )
IW pigments (mg m )
5000
6
0.15 3500
500
0
J FMAMJ J A S ON D
0
0
2.5
J FMAMJ J A S ON D
0
0.7
1500
0
J FMAMJ J A S ON D
0
3.5
7000
0
J FMAMJ J A S ON D
0
F 3. The seasonal distribution of pigments in the three layers of the interstitial water/sediment system (IW: 0–3 mm, 10–13 mm, 30–33 mm) and sea water (SW: 0–8 m). 30–33 mm; 10–13 mm; 0–3 mm; — — SW.
signatures or biomarkers) and these included Chl a as an indicator of total phytoplankton biomass, Per (dinoflagellates), Fuco (diatoms), HEX (prymnesiophytes), and Zea (blue-green algae). However, the lack of specificity of some markers e.g. Fuco reduced the taxonomic precision from the pigment approach (Roy et al., 1996). It is worth noticing that the chromatogram [Figure 2(c)] of the trays before immersion (defaunated sand) showed a variety of pigments which might be produced from algae or bacteria that could survive on the dry sand. The seasonal distribution of pigments in the three interstitial water/sediment layers (IW: 0–3 mm, 10– 13 mm, 30–33 mm) and the sea water (SW: 0–8 m) are given in Figure 3. In SW all pigments showed a major peak in September and several minor peaks in various seasons without presenting a uniform seasonality. In September the pigment concentrations were very high (Chl a: 4·87 mg m 3; Fuco: 4·35 mg m 3). A minor peak of Chl a (0·67 mg m 3) coincided with a minor peak of HEX (0·26 mg m 3) in April. In July a minor Chl c peak was observed (0·12 mg m 3) and later in October two other peaks were recorded (Zea: 0·18 mg m 3, Pheo a: 0·74 mg m 3). Chl a, Pheo a and Fuco values were high throughout the year while BUF, Per and Chl b values were low. A marked seasonal variation of total pigment concentrations in IW (0–33 mm) was also recorded without a defined profile (Figure 3). Total maxima were found during the period April–May in the pigments: BUF (891 mg m 3 ), HEX (3035 mg m 3), Chl b
(1198 mg m 3), Pheo a (12586 mg m 3), during July–August in: Chl a (8378 mg m 3), Chl c (1052 mg m 3) Fuco (6202 mg m 3) and during November–December in Per (896 mg m 3) and Zea (519 mg m 3). The vertical distributions of various pigments in the three layers fluctuated on a monthly basis giving various patterns. Only in a few cases were certain pigments (Chl a, Chl c, Fuco and HEX) equally distributed in the three layers (Chl c: June–July, Fuco: January–December, HEX: March, Chl a: April–May– January) while the distribution between layers was unequal during the rest of the year. In a few exceptional cases, an unusually high accumulation was registered. For example in May, Per (peridinin) concentration reached up to 77% at the deeper layer whereas in summer it was accumulated (80%) at the top (Figure 3). Pigment annual concentrations, their ranges and the percentage relationship of the pigments among layers are given in Table 3. When the annual average percentages at the three layers were evaluated only HEX showed almost equal percentages (31%–34%– 35%) from the top to the deeper layer. On the other hand, some pigments presented a sharp decline from the top to the deeper layer (Pheo a: 51%–32%–17%, Zea: 40%–33%–27%, Chl b: 46%–37%–23%), or a minor decline (BUF: 38%–37%–25% , Chl a: 39%– 32%–30%). Chl c and Fuco exhibited an increase from the top layer to the deeper one (Chl c: 31%– 30%–38%, Fuco: 32%–30%–38%) whereas Per
1983 (951–3481) 39% 1606 (665–2520) 32% 1538 (670–2984) 30%
51271904 1709240 1025 238–1637 0·76 (0·25–4·87)
Intermediate layer (10–13 mm) Mean (mg m 3) Range Percentages
Deeper layer (30–33 mm) Mean (mg m 3) Range Percentages
IW (0–33 mm) Sum of three layers Mean (mg m 3)SD
Average of three layers Mean (mg m 3)SD
Trays Mean (mg m 3) Range
Sea water (0–8 m) Mean (mg m 3) SD Range
Chl a
Top layer (0–3 mm) Mean (mg m 3) Range Percentages
Pigments
0·16 (0·03–1·21)
212 24–356
20318
609332
223 (51–429) 38%
189 (36–334) 30%
196 (70–419) 31%
Chl c
0·05 (0–0·2)
37 0–91
12420
362344
107 (0–615) 23%
120 (0–312) 37%
146 (0–363) 46%
Chl b
0·08 (0·01–0·35)
129 40–200
10223
306151
81 (23–172) 27%
98 (45–236) 33%
126 (23–258) 40%
Zea
(0–0·02)
10 0–20
7238
197254
39 (0–206) 25%
62 (0–183) 37%
114 (0–788) 38%
BUF
0·17 (0·01–1·35)
384 45–648
424106
1228745
530 (60–1617) 35%
317 (135–651) 34%
425 (0–1063) 31%
HEX
0·03 (0–0·29)
270 53–480
11571
344312
121 (0–779) 34%
40 (0–203) 21%
183 (0–576) 45%
PER
0·82 (0–6·59)
402 0–714
1445571
43363273
897 (0–4124) 17%
1403 (241–3391) 32%
2031 (906–5072) 51%
Pheo a
0·49 (0·02–4·35)
1268 137–2320
1183144
35491362
1344 (837–1954) 38%
1066 (433–1687) 30%
1139 (338–2561) 32%
Fuco
T 3. Pigment annual means, ranges and percentages (%) in interstitial water/sediment system (IW) and annual means and ranges in sea water (SW) and trays. Data are averages from the two stations S1 and S2
Algal pigments of an Eastern Mediterranean coastal area 421
422 A. Metaxatos & L. Ignatiades T 4. Pigment ratios in sea water (SW) and interstitial water/sediment (IW) Pigment ratios Medium
Fuco/Chl a
Chl c/Chl a
HEX/Chl a
SW
IW
SW
IW
SW
IW
SW
IW
SW
IW
SW
IW
January February March April May June July August September October November December Mean
0·26 0·56 0·09 0·16 0·39 0·16 0·26 0·63 0·89 0·27 1·09 0·28 0·42
0·67 0·71 0·77 0·66 0·36 0·56 0·54 0·76 0·99 0·80 0·90 0·82 0·81
0·09 0·19 0·22 0·12 0·13 0·15 0·43 0·25 0·25 0·10 0·24 0·09 0·19
0·09 0·13 0·10 0·19 0·05 0·15 0·13 0·14 0·18 0·21 0·09 0·03 0·12
0·11 0·19 0·04 0·39 0·08 0·22 0·15 0·15 0·28 0·12 0·03 0·21 0·17
0·05 0·13 0·31 0·57 0·20 0·34 0·18 0·17 0·30 0·41 0·40 0·05 0·26
0·01 <0·01 <0·01 0·03 <0·01 <0·01 <0·01 <0·01 <0·01 0·01 0·02 <0·01 0·01
<0·01 <0·01 0·03 0·17 0·02 0·03 0·04 0·04 0·08 0·02 0·02 <0·01 0·04
0·16 0·08 0·05 <0·01 0·04 0·05 0·05 0·08 0·04 0·10 0·10 0·43 0·10
<0·01 0·01 0·05 0·15 0·17 0·11 0·07 0·03 0·03 0·08 0·09 0·04 0·07
0·28 0·05 <0·01 0·71 0·19 1·17 0·81 1·25 1·35 2·17 0·35 1·47 0·82
0·36 0·36 1·07 1·59 1·82 0·80 0·27 0·30 0·61 1·60 0·85 1·04 0·89
showed a different pattern since its annual mean percentage was higher at the top (45%), that at the deeper layer was next (34%) and that in the intermediate layer was lower (21%). The pigment/Chl a concentration ratios in the sea water (SW) and the interstitial water/sediment layers (0–33 mm) from all sampling periods are shown in Table 4. The ratio is a convenient index of the relative concentration of both pigments, reducing the variance between consecutive chromatographic analyses when different samples are compared (Vernet & Lorenzen, 1987). These ratios displayed a variability throughout the year in both media, IW and SW, without a distinct profile. In SW ratios ranged from <0·01 to 2·17 and in IW from <0·01 to 1·82. High ratios were encountered in SW and IW during autumn (SW: Fuco/Chl a: 1·09; Pheo a/Chl a: 2·17 and IW: Fuco/Chl a: 0·99; Chl c/ Chl a: 0·21) and certain ratios exhibited their maxima during spring (HEX/Chl a=0·57, BUF/Chl a=0·17, Pheo a/Chl a=1·82 and Chl b/Chl a=0·17) only in IW. Figure 4 focuses on the seasonality of selected pigments (Chl a, Chl c, HEX, Zea, Pheo a, Fuco) in the interface of the two media e.g. the near bottom water (NBW) and the top layer (0–3 mm) of interstitial water/sediment system by using the mean values of the two stations S1 and S2. Fuco showed a peak in the top layer of IW in August (2560 mg m 3) followed by a NBW peak in September (4·1 mg m 3) and a similar pattern followed by Chl c and Chl a. Pheo a gave two different peaks in May in the top layer (5072 mg m 3) and a second in September in NBW (7·2 mg m 3) without any time coincidence. According to Figures 3 and 4 two general features were identified: the existence of a time lag between the formation of pigment peaks (with the IW peak
Buf/Chl a
Chl b/Chl a
Phaeo a/Chl a
being always first), and a difference in the seasonal distribution of peaks among pigments. The multiple regression analysis could estimate and define the quantitative relationships by revealing statistically significant differences in pigment contributions to Chl a biomass among various layers of IW or depths in SW and between media which might not be defined by ratios or correlations. According to this analysis (Table 5) Fuco exhibited a strong contribution with Chl a biomass in all layers of IW. In the top layer (0–3 mm) HEX (a marker for the water column phytoplankton) had a negative relationship with Chl a. In the intermediate layer (10–13 mm) Chl b had a positive relationship while Zea had a negative relationship to Chl a biomass. It is worth noticing that in the deeper layer (30–33 mm) a positive relationship of BUF and Pheo a with Chl a were found. This analysis also gave a positive relationship of Chl a with Chl c in SW and with HEX in NBW. On the other hand, the Pearson’s correlation analysis showed no significant correlation among pigments in the SW and IW. Discussion The present investigation focused on the estimation of various critical parameters in the interstitial water (IW) and water column (SW) systems affecting the pigment seasonal distribution in both media. The values of physicochemical parameters (Tables 1 and 2) were similar at the two stations S1, and S2, and according to the nutrient levels in SW (N-NO3: 0·07– 6·1 g-at; P-PO4: 0·04–4·1 g-at) the area could be characterized as inshore eutrophic (Ignatiades et al., 1992). Pigments in the IW can be derived from
Algal pigments of an Eastern Mediterranean coastal area 423 4.5
4500
1
450
Chl a
Chl c 3
3000
300 0.5
1.5
–3
0
150
0
1200
1.2
0
0
300
HEX
0.4 Zea
800
200 0.6
400
0.2 100
0
3000
4.5
0
0
6000
Fuco
–3
0
Pigments of NBW (mg m )
Pigments of IW (TL) top layer (mg m )
1500
8 Pheo a
2000
3
4000 4
1000
1.5
0
0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
0
2000
F 4. Seasonal variation of pigments in the top layer of IW (0–3 mm) and near bottom water (NBW).
NBW;
TL.
T 5. Parameters of the multiple regression equations for the SW (0–8 m), NBW, IW (0–33 mm) and the three layers of IW. Only the significant (P<0·05) beta coefficients () have been given Beta coefficients Source SW (0–8 m) NBW IW (0–33 mm) IW:TL (0–3 mm) IW:IL (10–13 mm) IW:DL (30–33 mm)
Fuco
HEX
Chl c
Chl b
Zea
BUF
Pheo a
Intercept
R2
P
0·694
0·059 0·278 136·100 455·100 685·300 65·000
0·988 0·988 0·710 0·960 0·950 0·820
0·000 0·000 0·000 0·001 0·016 0·010
0·975 0·668 0·822 0·836 0·478
0·861 0·330 0·710 0·739
microphytobenthos grown in situ, macroalgae detritus and sedimented phytoplankton and tychoplankton (Lucas & Holligan, 1999). The sandy bottoms of the two stations S1 and S2 had a similar grain size distribution with the fraction of grains (>0·5 m) being about 57% (Table 1). The trays had a coarser deposit with this fraction being greater than 75·5%. Although the defaunated sand
0·490 0·701
filling the trays before immersion was collected from the seabed of the sampling stations, a different grain sorting was produced gradually by the water movement in their restricted volume in relation to seabed (Table 1). Thus, the different levels of pigments (Table 3) recorded in the trays can be related to their different grain size distribution. Snelgrove and Butman (1994) suggest that grain size covaries with
424 A. Metaxatos & L. Ignatiades
porewater chemistry and microbial abundance and composition which could directly or indirectly influence infaunal populations. In the literature there is a contradiction regarding the relationship between the type of sediment and its composition of algae. Lucas and Holligan (1999) found greater Chl a in siltier sites than sandier ones and they also recorded a positive correlation between Chl a and silt. Furthermore, these authors reported that on the Molenplaat tidal flat (SW Netherlands) Fuco/Chl a ratios (0·35–1·60) indicated the dominance of diatoms and that the distribution of Chl a, Fuco and other pigments were correlated with grain size of sediments. However, in Port Phillip bay (S. Australia) no significant differences were found in Pheo a and Chl a values and ratio of total organic matter to Chl a across various sediment types (Light & Beardall, 1998). One explanation for these inconsistencies is that grain size is not an adequate descriptor of the sedimentary environment. Thus, the water flow on the tray surface (the trays protruded about 10 cm from the surrounding bottom) might be different from the encircling seabed altering the interfacial transport of algal cells into sediment. In addition to their taxonomic role, pigments can provide information on the physiological condition of algae (Hallegraeff & Jeffrey, 1984) and on the fate of these cells (Barlow et. al., 1995). More specifically, Chl a is commonly used as an indicator of phytoplankton biomass while the concentrations of its degradation products (for example Pheo a) are used as diagnostic indicators of physiological status, detrital content and grazing processes in natural phytoplankton communities (Mantoura & Llewellyn, 1983). There is no previous mention in the literature for pigment seasonality in the IW and the available information on the pigment distribution in sediment on a monthly basis is scarce (Abele, 1991; Sun et al., 1994). In the present investigation the temporal variations of IW pigments did not follow their seasonal variation in SW since in SW (0–8 m) and NBW (Figures 3 and 4) all pigments showed a peak in September but in IW the pigments displayed a various seasonality without a defined profile, resulting in the lack of any significant correlation between the two media. Also, there seemed to be a time lag between maximum values in SW and IW with the peaks in IW preceding those in SW (Figure 3). A similar conclusion has been reported by Poister et al. (1999) who found that the period of increased chlorophyll concentration in the water column corresponded to a period of decreased chlorophyll sedimentation rate and changes in the rates of pigment sedimentation did not reflect changes in pigment concentration in the water column of Trout Lake (Wisconsin, USA). It was
also reported (Rodriguez & Guerrero, 1994) that in Malaga Bay no clear temporal correlation between the water and the sediment pigment concentrations could be seen, even when the time-lag of water/sediment interface processes was considered. Special emphasis was given in the analysis of data derived from the top layer of IW and NBW e.g. the interface of two media. The pigment variation in NBW and top layer (TL, IW) gave a variety of patterns (Figure 4). NBW however, was not only affected by fluxes from beneath through TL but also from settling material from the water column which was mixed with resuspended particulate matter. In shallow waters wave action and currents will often be the dominating factors for particle fluxes across the interface (Graf & Rosenberg, 1997). Factors such as lateral redistribution of particles and different degradation rates between pigments may alter any simple correlation of water column and IW composition (no significant correlation between pigments found in our analysis) and therefore any similarity of pigment seasonality. This sound difference in temporal and spatial pigment fluctuation and magnitude between the two systems SW and IW can be additionally related to benthic in and epifauna activities. Gravitational settling, filtration activities of benthic animals and aggregation at the sediment–water interface withdraw particles from the NBW (Huettel et al., 1996). Fuco was the most abundant accessory pigment in all media giving a total average 0·49 mg m 3 in SW, 1183 mg m 3 in IW and 1268 mg m 3 in trays (Table 3, Figures 3 and 4). This can be due to an accumulation of benthic and pelagic diatoms on this sandy site, although their origin as benthic or pelagic can not be identified precisely. Our results support the aspect that coarse sandy sediment in shallow waters provides ideal substrate for benthic diatoms rich in Fuco (Abele, 1991) and that diatoms generally contribute a greater proportion of their biomass to sediments relative to other phytoplankton (Reynolds et al., 1982). Furthermore, Fuco was also found as the dominant carotenoid pigment of deposits in various depths of Cretan Sea (Duineveld et al., 2000) as well as in silty and sandy sediments in Westerschelde estuary (Lucas & Holligan, 1999). According to Bianchi et al. (1993) the benthic diatoms are considered as high quality (low C/N ratio) living resources and one of the most nutritious foods for marine and aquatic invertebrates. Pheo a was found in most samples and it was an abundant pigment in both media reaching very high values (6·59 mg m 3 in SW and 5072 mg m 3 in IW). The levels of this pigment tended to elevate in IW in spring when Chl a decreased and benthic macrofaunal activity was high.
Algal pigments of an Eastern Mediterranean coastal area 425
Light and Beardall (1998) found in Port Phillip bay that significantly greater Pheo a was associated with shallow sites and the uppermost sediment stratum than in deep water sites and lower strata. As far as vertical distribution of pigments in IW layers is concerned it was shown (Table 3) that Chl a (39% TL, 32% IL, 30% DL) profiles exhibited a slight decrease with depth from TL (0–3 mm) to DL (30–33 mm) contrasting the results of MacIntyre and Cullen (1995) and Lucas and Holligan (1999) who also examined the vertical profiles in the upper (0–3 cm) sediment layers and found a rapid decrease of Chl a over the top 1 cm. Also, Sun et al. (1994) found a nearly exponential decrease of Chl a within the upper 4 cm in Long Island. The profiles of Chl b (46% TL, 37% IL, 23% DL), BUF (38% TL, 37% IL, 25% DL) and Pheo a (51% TL, 32% IL, 17% DL) showed a sharper decline but Fuco (32% TL, 30% IL, 38% DL) and Chl c (31% TL, 30% IL, 38% DL) exhibited a slight increase with depth. The penetration distribution changes on a monthly basis (Figure 3) exhibited some extreme accumulations in the top (Chl c: May; Per: June, July, August, November) or in the deeper layer (Pheo a: January, February; Per: May; Chl c: June, July). According to Lucas and Holligan (1999) the deposition of pigments in the sediment is a complex phenomenon. For example, the sinking-sedimented phytoplankton such as dinoflagellates (Per) or prymnesiophytes (HEX) tended to be accumulated in the top layer (Figure 3) and the observed from time to time high levels of peridinin in the intermediate and deeper layer might be due to various reasons e.g. the vertical migration (phototaxis), permeability of the sediments and bottom currents, but there no available data for these parameters. On the other hand, Fuco, Chl a and Chl b are short lived markers associated with recent phytoplankton sedimentation events (Abele, 1991) decreasing sharply with depth. Factors like sediment type, depth, resuspension, grazing, as well as degradation rates, transformations in situ and half-life of pigments seem to be critical for the temporal and spatial distribution of pigments in the sediments. Interpretation of pigment ratios (Table 4) must be carried out with a degree of caution because this approach lacks statistical validation. Nevertheless, we estimated the ratios of six pigments and we draw some useful conclusions e.g. the phaeo a/Chl a ratios provide an indication of the physiological state of benthic microalgal population, where high values correspond to stressed or declining populations as mentioned by Light and Beardall (1998). In the present study, the major pigment peaks were recorded in SW in September and they were followed by a very high ratio
Pheo a/Chl a (SW: 2·17; IW: 1·6) in both media that could be associated with the declining populations in October. Also, the high Fuco/Chl a ratios in both media (SW: 0·42, IW: 0·81) of our area might reflect the dominance of diatoms whereas the low BUF/Chl a and Chl b/Chl a ratios might show the shortage of the taxa having the relevant pigments. The pigment analysis data of this work demonstrate that the algal communities thriving in the strongly interacting sea water and interstitial water/sediment system have a differing seasonally quantitative and qualitative composition. This might be attributed to the action of the sand bed as a gigantic filter that accumulates the algal cells forming thus a distinct community structure.
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