The methodological discussion regarding HPLC pigment analysis on microalgal samples is still object of debate in the literature (Buffan-Dubau and Carman, 2000; Latasa et al.,2001). Presently, the establishment of a universal methodology has now been abandoned in favor of the need to adapt it to the characteristics of the samples. This paper describes a method that gives good peak discrimination in microphytobenthos samples, and identifies without doubt the presence of degraded products of chloropigments by the simultaneous use of two detectors, the fluorimeter and the photodiode array. The advantage of this double detection had already been shown by us in previous works (Brotas and Plante Cuny, 1996; Brotas and Plante-Cuny, 1998), as well as by other authors studying benthic microalgae (Cariou-Le Gall and Blanchard; Klein and Riaux-Gobin, 1991). Prior to the analysis of microphytobenthos samples, peak identification in chromatograms of monospecific cultures from the algal classes expected in the community, is a valuable help. Nonetheless, care must be taken when designating marker pigments for algal types based on the examination of restricted algal species within each group (Jeffrey, 1997), as there is some diversity in pigmentation within taxonomic groups. Particularly within the class Bacillariophyceae, Pennington et al. (1998), analyzing 10 different species, concluded that b-b-carotene, diatoxanthin, diadinoxanthin and fucoxanthin were always present, whereas fucoxanthinol was found in four species. Results from Fig. 1 were checked in the literature (Jeffrey et al., 1997;Wright et al., 1991); all species studied presented typical pigment signatures of their respective taxonomic class, except for G. instriatum, whichis a cosmopolitan species, common in estuarine and neritic waters. Peak no. 3 was identified as chlorophyll c2 by comparison of spectral characteristics in the eluant with Kraay et al. (1992), on the other hand, a compound possibly corresponding to chlorophyll c1 eluted just before chlorophyllide a in the diatom culture and in some sediment samples, however, as its concentration wasminute, further experiments are needed to check this identification.Previous works on Tagus estuary mudflats indicated that microphytobenthos is constituted by diatoms (the dominant group), euglenophytes, cyanobacteria and possibly cryptophytes, whilst dinoflagellate species were never spotted in microscopic observations (Brotas and Catarino,1995; Brotas and Plante-Cuny, 1998; Riberio et al., 2003, this volume).The need for information regarding pigment composition and pigment ratios of microalgal taxonomic groups is continuously stressed in the literature. Pigment ratios displayed in Table 3 arewithin the range of the ratios presented by other authors (Desey et al., 2000; Mackey et al., 1997; Schlüter et al., 2000). The ratio of diatoxanthin/Chla presents the highest variation between classes (from0.299 in euglenophytes to 0.004 in low light diatoms), and suffers the most pronounced increase in response to light intensity. Discussing the effect of light on pigment ratios, Schlüter et al. (2000) report that the ratios of the non-diagnosticant pigments diadinoxanthin,diatoxanthin and b-carotene to Chl a were strongly influenced by light intensity, increasing markedly, whereas the ratio of diagnostic pigments such as fucoxanthin (for diatoms) and chlorophyll b (for euglenophytes) showed minor variations. Nevertheless, numerous authors assume that pigment ratios do not change through a data set, enabling the estimation of the contribution to each taxonomic group to the total phytoplankton biomass ((Barlow et al.,1993; Desey et al., 2000; Gieskes and Kraay, 1983), amongst others).
Figs. 2 and 3 illustrate examples of pigment compositionon surface sediments and depth distribution in intertidal mudflat sites. The importance of pheophorbides a as the major degradation product of chlorophyll a agrees with previous works (Brotas and Plante-Cuny, 1996; Brotas and Plante-Cuny, 1998; Cariou-LeGall and Blanchard;Klein andRiaux-Gobin, 1991). As the typical set of carotenoids for diatoms, fucoxanthin, diatoxanthin, diadinoxanthin and b-b-carotene (Pennington et al., 1998), is constant in all samples,the detection of other pigments, even in trace quantities, is fundamental to indicate the occurrence of other taxonomic groups.Considering only the case of fucoxanthin, the biomarkerfor diatoms, certainly themost abundant carotenoid in microphytobenthic samples, the ratio Fucoxanthin/Chla obtained on the upper sediment layer was between 0.295 and 0.486,decreasing markedly with depth. This result suggests thatfucoxanthin has higher degradation rates than chlorophyll a,as already pointed out by Cariou-Le Gall and Blanchard(1995). In order to discuss the ecological implications of these results, a greater number of samples are needed; this will be done in future works. The present paper pretends essentially to be a methodological contribution to HPLC analysis of photosynthetic pigments.
In conclusion, one of the main goals of HPLC pigment analysis is the reduction of the time-consuming microscopic observations. Within a given set of samples, providing a certain number of samples is checked through both techniques, pigment analysis can evaluate spatial or seasonal distribution of microphytobenthos biomass and composition through the quantification of chlorophyll a and pigment biomarkers. Moreover, relevant aspects of the ecology of coastal sediments can be revealed as a result of this approach;the evidence of plant detritus suggests that a studied site is located on an area where accumulation of allochthonous material occurs (Abele-Oeschger, 1991), the abundance of pheophorbides a is related to a high grazing activity (Bianchiet al., 1993; Brotas and Plante-Cuny, 1998, Mackey et al.,1996).
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Figs. 2 and 3 illustrate examples of pigment compositionon surface sediments and depth distribution in intertidal mudflat sites. The importance of pheophorbides a as the major degradation product of chlorophyll a agrees with previous works (Brotas and Plante-Cuny, 1996; Brotas and Plante-Cuny, 1998; Cariou-LeGall and Blanchard;Klein andRiaux-Gobin, 1991). As the typical set of carotenoids for diatoms, fucoxanthin, diatoxanthin, diadinoxanthin and b-b-carotene (Pennington et al., 1998), is constant in all samples,the detection of other pigments, even in trace quantities, is fundamental to indicate the occurrence of other taxonomic groups.Considering only the case of fucoxanthin, the biomarkerfor diatoms, certainly themost abundant carotenoid in microphytobenthic samples, the ratio Fucoxanthin/Chla obtained on the upper sediment layer was between 0.295 and 0.486,decreasing markedly with depth. This result suggests thatfucoxanthin has higher degradation rates than chlorophyll a,as already pointed out by Cariou-Le Gall and Blanchard(1995). In order to discuss the ecological implications of these results, a greater number of samples are needed; this will be done in future works. The present paper pretends essentially to be a methodological contribution to HPLC analysis of photosynthetic pigments.
In conclusion, one of the main goals of HPLC pigment analysis is the reduction of the time-consuming microscopic observations. Within a given set of samples, providing a certain number of samples is checked through both techniques, pigment analysis can evaluate spatial or seasonal distribution of microphytobenthos biomass and composition through the quantification of chlorophyll a and pigment biomarkers. Moreover, relevant aspects of the ecology of coastal sediments can be revealed as a result of this approach;the evidence of plant detritus suggests that a studied site is located on an area where accumulation of allochthonous material occurs (Abele-Oeschger, 1991), the abundance of pheophorbides a is related to a high grazing activity (Bianchiet al., 1993; Brotas and Plante-Cuny, 1998, Mackey et al.,1996).
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