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Fatty acids, chlorophylls and total silicon in mucilaginous aggregates collected in a coastal area of the Northern Adriatic Sea facing Emilia-Romagna in August 1988
The Science of the Total Environment 165 (1995) 193-201
Fatty acids, chlorophylls and total silicon in mucilaginous aggregates collected in a coastal area of the Northern Adriatic Sea facing Emilia-Romagna in August 1988 R. Viviani*“, L. Boni”, 0. Cattania, A. Milandrib, M. Pirini”, R. Polettib, M. Pompeib “Centro
di ricerca interdipartimentale bConsoRio di studi, ricerche
de& scienze dei mare, Uniwrsitli di Bologna, Bologna, e interventi sulle risorse marine di Cesenatico, Forli Italy
Italy
Abstract
Fatty acid, chlorophylls a&c and total silicon analysis were carried out in mucilaginous aggregates collected in a coastal area of the Northern Adriatic Sea facing Emilia-Romagna in August 1988, with the aim of contributing to determine the taxonomy of the producers. The combined data seem to con&m the idea that the mucilaginous aggregates mostly derive from the diatoms responsible for typical blooms in the Adriatic sea. When the presence and morphology
of the organisms
in the mucilaginous
aggregatesis not clear, the biochemicalmarker study seemsto be
useful not only for taxonomic determination of marine micro and macro algae and other eukariotic organisms, but alsofor marine prokariotes. Keywords:
Mucilaginous aggregates; Adriatic Sea; Microalgae; Macroalgae; Diatoms; Dinoflagellates; Phytoplankton
1. Introduction
During July and August 1988, a ‘dirty sea’ phenomenon characterized by very large quantities of mucilaginous aggregates not previously observed in the last 40 years, was recorded along the coasts of the northern and middle Adriatic (Brambati, 1988; De Gobbis, 1989). The first signs occurred at the end of June at Lussino and on 26 July at Rovigno (Bressan,
* Corresponding author. 004%9697/95/$09.50 SSDZ
1988). The collection and studies of mucilaginous aggregates were carried out by the laboratories of Portorose, Trieste, Venice, Cesenatico, Fano and Ancona. As the phenomenon of ‘dirty water’ has been connected with diatoms (since the last century), studies have been directed towards pelagic and benthonic diatoms. At Portorose in the ‘marine snow’ accumulated in the south-east area of the gulf of Trieste, Malej and Faganeli (1988) noted diatoms, the most prevalent being Nitzschiu closterium, Leptocylindrus dunicus, Chaetoceros simplex, and Skeletonema costatum.
0 1995 Elsevier Science BV. All rights resewed.
0048-9697(95)04552-X
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R. Vkiani et al. /Science of the Total Environment 165 (1995) 193-201
According to Fonda Umani et al. (1988) at Trieste, few animal organisms and phytoplankton cells were evident in the material collected on 13 August 1988, while on 16 August, there were enormous quantities of pelagic diatoms (Nitzschiu, spp., Leptocylindms sp., Chaetoceros spp., and Skeletonemu co.stutum). From 17 August onwards, the same quantities of diatoms were no longer present in the mucilaginous aggregates. In the Venice laboratories, the examination of the material indicated the highest abundance of the diatom Nitzschia closferium (France, 1988). During the monitoring programme of eutrophication along the Emilia Romagna coast in the mucilaginous aggregates collected for the first time on 8 August at Ravenna and examined in the Cesenatico laboratories, it has been demonstrated that the dominant species of diatoms was Nitzxhia clostetium and, among the dinoflagellates, Gonyuular frugilis. On the same day, in material collected at Punta Marina, the dominant diatoms were Skeletonema costatum, Nittschia closterium, and lrhalassiosirasp., while the dominant species of dinoflagellates were Gonyaz& polyedru and Oxytoxum Zongiceps(Consorzio Cesenatico, 1988). At the beginning of August, at the same time as the mucilaginous aggregates appeared, there was a bloom of phytoflagellates probably Chrysophyceae (14 million cells per I) at Port0 Garibaldi. The mucilaginous aggregates collected on 12 August 1988 at Marina Romea had Nitzschia closterium and Thalassiosira sp. as dominant diatoms and for the dinoflagellates Gonyuuluxfiagilis, while those on 16 August collected at Bellaria contained Nitzschia closterium and Gonyaulax frugi1i.s in addition to fragments of macroalgae (Consorzio Cesenatico, 1988). We have attempted a biochemical taxonomic approach with the aim of contributing to the knowledge of algal organisms from which the first mucilaginous aggregates formed, and which subsequently included other micro- and macroalgae (and also some microzooplankton and micrometazoan organisms), and which were subjected to the action of marine bacteria. In this report, we have evaluated whether it could be useful to take into consideration the
pattern of fatty acids of total lipids, chlorophylls a, b, c and total silicon analysis of mucilaginous aggregates in order to ‘finger print’ the material with regard to its origin and to contribute to the knowledge of the molecular nature of mucilaginous aggregates in their tinal stages. Following the suggestion by Hildich (1940) on the role of lipid constituents on quantitative chemical taxonomy, a voluminous literature on fatty acids composition has been generated for use in the description and classification of species (Holman, 1978). Various investigations have revealed that the fatty acid composition of lipids are chemotaxonomic markers useful not only in the classification of micro- and macroalgae (Parker et al., 1967; A&man et al., 1968; Parsons and Takahashi, 1975; Pohl and Zurheide, 1979; Harwood, 1988; Harwood et al., 1988) (Table 11, but also for prokariotes (Cyanobacteria and Eubacteria) (Parsons and Takahashi, 1975; Schleifer and Stackebrandt, 1983; Fulco, 1983; Harwood et al., 1988; Rajendran et al., 1992). Also, chlorophylls u, b, c and total silicon could give additional taxonomic indications for microand macroalgae. Cyanophyceae have chlorophyll a only, Chlorophyceae a and b, Bacillariophyceae (Diatoms) and Dinophyceae (Dinoflagellates) a and c. Silicon is present in the frustule of diatoms as hydrated amorphous silica (SiO,*nH,O), but it is absent in other algae. In this report, fatty acids (Table 2) of total lipids (Table 3) were determined in a sample of mucilaginous aggregate, collected on the sea surface on 16 August 1988, about 2 km off the west coast of Bellaria, at the central point of the Emilia-Romagna coast. In order to have additional taxonomic indications, chlorophylls a, b, c, and total silicon (Table 3) were analyzed on a dry weight basis. To evaluate the chemical and biochemical data obtained from samples of mucilaginous aggregates in relation to the species of phytoplankton present in the sea water column in August and in the preceding periods, data on phytoplankton blooms in the area of the sea off the coast of Emilia-Romagna from the months of June-August 1988 (Table 4) have been reported. Of the blooms found, only the most abundant
17.2 -
9.4 -
0.6 2.6 2.5 -
3.7 -
1.8
0.4 0.4 25.4 24.9
5.5
0.6 2.2
1.1
-
0.4 1.7
-
3.7
0.5 1.2 20.8 -
-
0.5 0.1
3.4 0.5
0.3
2.9 8.7 1.2
0.1
13.8 -
-
-
0.5
11.0 21.2
-
0.6 6.3
0.1
1.4 6.9 22.4 7.4 2.3 6.6
1.0
0.2 32.7
Bacillariophyceae (diatoms, phytoplankton) A B C
-
3.2
6.5 4.3 19.8 -
-
0.2 0.4
0.4 11.3 21.7 2.8 9.4 1.2 0.2 2.9 0.8 -
-
0.5 6.3
D
polyedra
N = Alexandrium
tamarense
1= Prorocentrum
Values are % of total fatty acids; tr, trace. A = Nitzschia closterium B - Skeletonema costatum
2o:o + 2O:l 20:2 20~3 20:4n-6 20~4 n-3 20:5n-3 22:0+ 22:1 22~4 22:s n-3 2216 n-3
l&O l&l 18:2 n-6 18~3 n-6 18:3 n-3 l&4 n-3
I6:4
12:o 14:o 14:l 150 16:0 16:l 16:2 16:3
Fatty acid
0.4 2.2
8.0
0.1
0.5
tr
5.5
16.4 -
-
-
tr tr
2.7
tr
30.1 38.3 4.4 2.7
-
-
C = Chaetoceros septentrionale micans J = Monocrysis lutheri
-
-
0.2 0.2
23.2 44.8 2.8 6.5 0.1 0.3 0.4 0.4 -
0.1 1.0
7.9
0.1
E 2
3 2
23
3
1 18
-
-
5 12 -
-
3 5 5
28 4 2 -
11 1 -
-
0.5
2.4 6.0
6.4
12.4
-
-
-
-
11.6
-
9.8 22.7
13.6
0.4
0.4 13.1
16.3 1.2 -
-
-
0.2 4.0 0.6 0.1
0.1
0.8 0.7
0.2 11.4 0.2 0.2 15.1 25.4 1.9 0.6 0.3 -
Chrysophyceae (phytoplankton)
9.3 1.6 0.4 16.7 24.2 0.1 0.2 0.2 0.5 0.7 2.1 0.6 0.5 2.2 0.4
0.1
17.9 2.1 0.3 1.3 17.8
-
0.2 0.4
tr
2.3 0.7 0.8 2.6
1.4 1.0
20.8 16.9 0.5 0.2 0.4
0.7 9.4 5.6 0.6
12.5 2.6 0.8 2.9 15.7
-
0.8
0.1
Chlorophyceae (macroscopic) L
tr
0.5
0.5 0.4
45.5 -
-
0.8 0.5 1.3
1.0 16.1 1.7 -
-
18.2 3.4
-
1.9 8.1
Rhodophyceae (macroscopic) M
D = Asterionella japonica E = Thalussiosira fluvialis F = Navicula incerta G - Goniuular K = Ulva lactuca L - Enteromorpha intestinalti M = Gracilaria confervoides.
14 -
3 14 -
-
-
1
36
-
-
Dinophyceae (phytoplankton)
Table 1 Fatty acids composition (% of total fatty acids) of some genera and species of marine phytoplankton and marine macroalgae according to Pohl and Zurheide (1979)
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R Kviani et al. /Science of the Total Environment 165 (1995) 193-201
Table 2 Fatty acid composition of total lipids of ‘mucilaginous aggregates” Fatty acid
Weight %
120 14:Oi 14:o 150 16:Oi 16:0 16:l 17:o 17:l l&O l&l n-9 18:2 n-6 l&3 n-6 18:3 n-3 2o:o 2O:l n-9 l&4 n-3 21:o 20~4 n-6 220 221 n-9 20:5 n-3 240 241 n-9 22:5 n-3 22~6 n-3
1.5 f 0.0 0.3 f 0.0 10.0 f 0.9 1.0 f 0.1 0.2 f 0.0 31.4 f 1.0 20.1 & 1.5 0.5 f 0.0 0.2 f 0.0 4.1 f 0.2 13.0 f 0.6 3.4 f 0.3 0.4 f 0.0 0.7 f 0.0 0.3 f 0.0 0.8 f 0.0 0.3 f 0.0 Traces 1.0 f 0.1 0.4 f 0.0 4.2 f 0.4 4.3 f 0.0 0.4 f 0.0 Traces 0.4 f 0.0 1.1 f 0.1
‘Results expressed as the mean of duplicate determinations f S.E.
Table 3 Total lipids, silicon and phytopigments of ‘mucilaginous aggregates’ expressed as dry weight g/loo g Total lipids Total silicon Chlorophyll a Chlorophyll b Chlorophyll c Pheopigments
mg/lOO g
1.100 6.450 3.520 0.437 6.800 2.720
and stored at -20°C until analysis. In addition after filtration, the material used for determination of total silicon has been treated with ultraturrax before analysis. For each sample, dry weight has been determined (105°C). Samples were used for qualitative determination of phytoplankton species and for macroscopic examination. Investigation of bacteria content and the presence of microzooplanktons and micrometazoan organisms was not carried out. This was the subject of research by other authors in the same period in a coastal area of the northern Adriatic Sea facing Emilia-Romagna (Aulicino et al., 1989) and in the Gulf of Trieste (Cabrini et al., 1987-88, 1989; Milani et al., 1987-88; Fonda Umani and Ghirardelli, 1989). 2.2. Algae in sea water and mucilaginous aggregates
have been reported, those with a value greater than 5 million cells per litre. 2. Materials and methods 2.1. Mucilaginous aggregates:samplepreparation
A sample of mucilaginous aggregate was collected on 16 August 1988 on the surface of the northern Adriatic sea, about 2 km off the coast of Bellaria. The samples were collected in a number of 500-ml polyethylene bottles, skimming the gelatinous component. From this material used in sample preparation for phytoplankton studies and determination of total lipids, fatty acids, pigments and dry weight, the water was filtered through a nylon net of about 20-pm mesh size. The residue (for biochemical analysis) was immediately frozen
For algal studies, the sea water and the material were preserved with Lugol’s solution and examined by Uterrnohl’s (1958) method. For taxonomical studies, the sample was examined with a common light microscope. The phytoplankton data in mucilaginous aggregates are only qualitative. In Table 4, quantitative data of phytoplankton blooms from January to August are reported. 2.3. Total lipids determination and fatty acids analyses
Lipids were extracted according to Folch et al. (1957) from the sample of mucilaginous aggregates. The solvent mixture used for extraction contained 0.01% (w/v) BHT as antioxidant. Total lipids (TL) were determined gravimetrically. The saponification of total lipids, the extraction of
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Table 4 Principal blooms registered in 1988, in the period preceding the appearance of the mucilaginous aggregates in the area of sea off the coast of Emilia-Romagna Month
Station
Phytoplankton
Cells/l
January February April May July August
Lido Adrian0 Lido Adrian0 Rimini Lido Adrian0 Bagni di Volano Port0 Garibaldi
Hemiaulus hauckii (diatom) Thalassiosira sp. (diatom) Skeletonemu costatum (diatom) Chaetoceros spp (diatom) Chaetoceros spp (diatom) Chrysophyceae
5658OW 7242000 50 122500 110500ooo 23 341000 14934875
fatty acids (FA) and the methylation using 14% BF, in methanol were carried out as described by Borgatti et al. (1982). Methyl esters of FA were analyzed by gas-liquid chromatography (GLC) and also by chromatography on silicic acid columns impregnated with 25% AgNO, and by catalytic hydrogenation. GLC was done on a Dani (Monza, Italy) 3600 gas-chromatograph, using a glass column (2 m x 4 m) filled with acid-washed Chromosorb W 60-80 mesh and 20% (w/w) diethylene-glycolsuccinate. The carrier gas was nitrogen. The GLC operating conditions and the methods of identification of FA were carried out as reported by Pagliarani et al. (1986). Methyl esters of FA separated by GLC are given as weight percentages of total FA in total lipids (Table 2). 2.4. Chlorophyll and pheopigments Chlorophylls a, b, c and pheopigments
have been extracted from the sample with 90% acetone and estimated spectrophotometrically according to the method of Strickland and Parsons (1968) (Table 3).
2.5. Total silicon
The samples have been analyzed for total silicon according to Koroleff (1983a,b) (Table 3). Since the presence not only of reactive silicate (the dissohed ions of the orthosilic acid) but also of inorganic and organic fractions of diatom frustules (cell wall) is possible in mucilaginous aggregates, the samples were treated with an alkaline persulphate solution to break down these silicon complexes and convert them to reactive silicate.
3. Results and discussion 3.1. Algae in mucilaginous aggregatesand in sea water
Observations under the optical microscope have demonstrated the presence of an extremely heterogeneous amorphous structure in the samples examined, in which the presence of algal cells of diatoms and dinoflagellates, and fragments of macroalgae can be noted. The qualitative study carried out on phytopiankton species showed the presence of Nitzschia clostetium and Gonyaulax fragilis, the first being clearly predominant. From the comparison between the phytoplankton population present in the sea water column with some species of dinoflagellates and diatoms present in mucilaginous aggregates, the following species were not found in the latter: Gonyaulax po(yedra and Thalassiosira sp. The species Nitzschia clostetium and Gonyaulax @gilis were found both in the sea water and the mucilaginous aggregates. From the data in Table 4, it can also be seen that the phytoplankton blooms of January-August were the following: Hemiuulus hauckii, Thalassiosira sp., Skeletonema costatum, Chaetoceros sp. and Chrysophyceae. 3.2. Total lipids
In the dry weight of the ‘material’, total lipids represent 1.1%. These lipids are constituents of cellular membranes of all algal cells and of the lipid body in some diatoms. Traces of lipids of eubacteria are present, possibly also of microzoo plankton and micrometazoan captured from sea water. The superficial total microzooplankton
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R Viuiani et al. /Science of the Total Environment 165 (1995) 193-201
population in August is characterized by population values of some hundreds of individuals per litre and micrometazoan of some 20 individuals per litre (Milani et al., 1987-1988). 3.3. Fatty acid in total lipids in mucilaginous aggregates
From Table 2, it can be seen that for the fatty acids in the total lipids, 14:0 is present at 9.3%, 16:0 at 29.1%, 16:l at 18.7%, 18:0 constitutes 3.8%, 18:l at 12.1% and Cl8 polyunsaturated at 4.6%; 20:4 n-6 constitutes 0.9%, 20:5 n-3 constitutes 4.09% and 22:6 n-3 constitutes 1.1%. Comparing these data with the quantities of fatty acids characteristic of some types of diatoms and of dinoflagellates presented in Table 1, it corresponds to those present in the mucilaginous aggregates examined. The high percentage of 16:0 and 16:1, the presence of 20:4 n-6, the level of 20:5 n-3, and the lack of 22:6 n-3 are characteristic of the fatty acid spectrum of the diatoms (Skeletonemu, Chaetoceros, Nitzschia) rather than that of the dinoflagellates (Gonyaulux sp.) taken into consideration. In these, in fact, the quantity of 16:l is low (l-4%), while 22:6 n-3 is present in relatively high quantities (18-23%). However, the characteristics noted by us can also be common to any species of Chrysophyceae. Since a bloom of an unidentified species of Chrysophyceae was recorded at the beginning of August 1988 in the area of sea between the mouth of the river PO and Ravenna, one cannot exclude the contribution of this species to the formation of the mucilaginous aggregates examined by us. As far as the levels of 18:l (12.1%) are concerned, they are higher than those reported for the same diatoms and dinoflagellates in Table 1; these could be derived from, as far as the algae components are concerned, the numerous macroalgae present in the coastal area of EmiliaRomagna, above all, Chlorophyceae, of the Ulna genus, and Rhodophyceae such as Graciliaria confervoides and Polysiphonia sertularioides of which minuscule fragments were incorporated in the mucilaginous matrix analyzed.
Nevertheless, even the bacterial component, expressed as CFU/lOO ml, which in the same mucilaginous aggregates is enriched with respect to the average values taken in the same zone in the absence of mucilaginous aggregates (Aulicino et al., 1989) could be the source of mono-unsaturated fatty acids, above all 18:1, which are generally present in high percentages in the eubacteria (Rajendran et al., 1992). The low levels of 14:0 iso (0.3%) and 16:0 iso (0.2%) in addition to 15:0 (l.O%), 17:0 (0.5%) and 17:1(0.2%) in the sample indicates that the quantity of Eubacteria present does not represent a significant contribution to the lipid total of the mucilaginous aggregates. In bacteria, the lipids of Archaebacteria are distinguished from those of Eubacteria by the absence of fatty acid glycerol ester lipids and by the presence of isoprenyl glycerol ether lipids (Schleifer and Stackebrandt, 1983; De Rosa and Gambacorta, 1988). Since fatty acids are not constituents of Archaebacteria lipids, the presence of these marine bacteria could not be excluded in the sample analyzed. Concerning eubacterial lipids normal-chain fatty acids, iso and anteiso branched chain fatty acids containing 14-17 carbon atoms are the dominant acyl components of glycerol esters. In effect, with the exception of Archaebacteria, all prokariotes and eukariotes synthesize fatty acids (Fulco, 1983). Since previous blooms of diatoms Skeletonema spp. and Chaetoceros spp. (Table 4) could be responsible for the origin of ‘marine snow’, C16, C18, C20, and C22 polyunsaturated fatty acids could have undergone chemical autoxidation in the presence of oxygen or bacterial metabolic modification, like for example biohydrogenation in an anaerobic environment. In addition, since primitive ‘marine snow’ and the final ‘mucilaginous aggregates’ could capture some microzooplankton and micrometazoan organisms, it is not possible to exclude the presence of their fatty acids. Nevertheless, their presence cannot be proven by particular fatty acids as is possible in the case of bacteria which possess uneven and branched fatty acids.
R Viviani et al. /Science of the Total Environment 165 (1995) 193-201
3.4. Chlorophyll
The content of chlorophylls, expressed in mg/lOO g dry wt., is as follows: chlorophyll a, 3.52 mg; chlorophyll b, 0.437 mg; chlorophyll c, 6.80 mg; pheopigments, 2.27 mg. As the highest levels of chlorophyll are those of a and c, this indicates that the material is derived from diatoms and dinoflagellates. The lower levels of chlorophyll b suggest that it originates from micro and macro Chlorophyceae and, perhaps from a particular Gymnodinium sp. Since Gymnodinium spp. were not present either in the mucilaginous aggregates, or in the previous blooms, the data confirm that chlorophyll b is derived from Chlorophyceae. 3.5. Total silicon
In the dry weight of the samples examined, total silicon represents 6.54%. These high levels of total silicon indicate the presence of diatom frustules, because the concentration of suspended silicon (reactive silicate) is very small in sea water, usually exceeding a few pmol/dm3 (Koroleff, 1983a). Also according to De Gobbis (19891, as appears from the SEM-micrograph of the mucilaginous aggregates collected along the coast of Istria in August 1988, diatom frustules dominated over biogenic remains. The data suggest that in the final stage of mucilaginous aggregates, when large amounts of diatom frustules are present, in addition to the alkaline persulfate method (Koroleff, 1983a), a carbon fusion method could also be utilized (Koroleff, 1983b). 4. Conclusion The data on fatty acids, chlorophylls a, b, c and total silicon combined with the information on the previous and present blooms of phytoplankton and macroalgae could contribute to the knowledge of the evolution of mucilaginous aggregates and their molecular nature in their final stages. These combined data seem to confirm the idea that the mucilaginous aggregates derive from the diatoms responsible for typical blooms in the
199
Adriatic Sea, and also to the presence of material derived from Chlorophyceae and Eubacteria. The fatty acid composition of total lipids of samples of mucilaginous aggregates indicates that in the final stage in which there is no demonstrable presence of alga responsible for the phenomenon, the composition of fatty acid in this material can have a taxonomic significance. Our data confirm the findings of other authors (Herndl and Peduzzi, 1988; Stachowitsch et al., 1990; Rinaldi et al., 1992) that planktonic diatoms, such as Skeletonema and Chaetoceros that make up a major fraction of the usual spring bloom of plankton are the main producers of mucilaginous aggregates in the Adriatic Sea rather than dinoflagellates. As far as the condition of phytoplankton in the preceding months of 1988 is concerned, in April, a bloom of Skeletonema costutum was observed with 50 million cells per litre at Rimini, while, in May, all along the coast of Emilia-Romagna a bloom of Chaetoceros spp. appeared which reached 110 million cells per litre of water at Lido Adrian0 (Table 4). Also, high levels of Chaetoceros spp. from Istria to the PO delta (increasingly so near the latter), was reported in May (De Gobbis, 1989). In June, a reduction occurred, while in July at Bagni di Volano, there was a peak reached of 23 million cells per litre. In August, the presence of Nitzschia clostetium was one of the main characteristics of the material collected in the high Adriatic Sea. As the succession of diatoms in bloom in the Northern Adriatic Sea usually starts with Skeletonema and is followed by Chaetoceros and Nitzschia, it will be necessary to evaluate the role of these diatoms at the beginning and in the various phases of the complex phenomenon, which leads from the ‘marine snow’ to the formation of the mucilaginous aggregates. It will also be interesting to evaluate the participation of other algae and of some dinoflagellates of the Gonyuulax genus and the action of marine prokariotes. In all phases of the research, when the morphology of the organisms is not clear, it could be useful, in the area of chemical and biochemical analysis, to examine the spectrum of the fatty
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R viviani et at. /Science of the Total Environment 165 (1995) 193-201
acids of the total lipids for a taxonomic contribution. In addition to the analysis of protein, carbohydrates, total lipids, ATP, phytopigments and steroids (these last two being recently indicated by UNESCO (1991) for the study of harmful algal blooms), fatty acids and other lipid constituents must also be taken into consideration in the monitoring of ‘marine snow’ and mucilaginous aggregates. This report is a methodological approach to studying in addition to fatty acids, other lipid constituents in various stages from ‘marine snow’ to ‘mucilaginous aggregates’ not only as biochemical taxonomic markers useful for marine micro and macro algae and other eukariotic organisms, but also for marine prokariotes. References A&man, R.G., C.S. Tocher and J. McLachlan, 1968. Marine phytoplankter fatty acids. J. Fish. Res. Board Can., 25: 1603-1620. Aulicino, F.A., L. Bonadonna, G. Bucci, M. de Mattia, I. di Girolamo, P. Maini, L. Mancini and L. Volterra, 1989. La componente microbiologica durante il fenomeno de1 mare sporco. Mare Adriatico: agosto 1988. Ing. San., 37: 23. Borgatti, A.R., G. Lenaz, A.M. Sechi, G. Trigari, A Pagliarani and V. Ventrella, 1982. Studies on lipid composition and physical state of liver and heart mitochondria and microsomes in pigs fed on diets containing dried biomass of Candida lipoZytica (Top&a). Food Sci., 47: 59-64. Brambati, A, 1988. Quale Adriatico? In: 11 fenomeno de1 mare sporco nell’Adriatico (luglio-agosto 19881, Consiglio Nazionale alle Ricerche, Roma-Trieste, pp. 3-6. Bressan, G., 1988. In: 11 fenomeno de1 mare sporco nell’Adriatico (luglio-agosto 1988), Consiglio Nazionale alle Ricerche, Roma-Trieste, pp. 3-6. Cabrini, M., L. Milani, G. Honsell and S. Fonda Umani, 1987-1988. The phytoplankton in a station in the Gulf of Trieste from March 1986 to September 1988: data report. Nova lbalassia, 9: 11-52. Cabrini, M., L. Milani, S. Fonda Umani and G. Honsell, 1989. Relazioni trofiche tra fitoplancton e microzooplancton nel Golf0 di Trieste. Oebalia, 15(l) N.S.: 383-395. Consorzio di studi, ricerche ed interventi sulle risorse marine di Cesenatico, 1988. Eutrofkzazione de1 litorale emilianoromagnolo. Rapport0 annuale per Regione EmiliaRomagna. Assessorato alla sanita. De Gobbis, D., 1989. Increased eutrophication of the Northern Adriatic Sea. Second Act. Mar. Pollut. Bull., 20: 452-457.
De Rosa, M. and A. Gambacorta, 1988. The lipids of archaebacteria. Prog. Lipid Res., 27: 153-175. Folch, J., M. Lees and G.H. Sloane-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem., 226: 497-509. Fonda Umani, S. and E. Ghirardelli, 1989. Caratteristiche chimiche e biologiche de1 sistema pelagico de1 Golfo di Trieste. Hydrores., 6: 71-82. Fonda Umani, S., E. Ghirardelli and M. Specchi, 1988. In: 11 fenomeno de1 mare sporco nell’Adriatico (luglio-agosto 19881, Consiglio Nazionale alle Ricerche, Roma-Trieste, pp. 37-42. France, P., 1988. In: 11 fenomeno de1 mare sporco nell’Adriatico (luglio-agosto 19881, Consiglio Nazionale alle Ricerche, Roma-Trieste, pp. 43-46. Fulco, A.J., 1983. Fatty acid metabolism in bacteria. Prog. Lipid Res., 22: 133-160. Harwood, J.L., 1988. Fatty acid metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol., 39: 101-138. Harwood, J.L., T.P. Pettitt and AL. Jones, 1988. Lipid metabolism. In: L.J. Rogers and J.R. Gallon (Eds.), The Biochemistry of the Algae and Cyanobacteria. Gxford University Press, London, pp. 40-67. Herndl, G.J. and P. Peduzzi, 1988. Ecology of amorphous aggregations (marine snow) in the Northern Adriatic Sea. I. General consideration. P.S.Z.N.I.: Mar. Ecol., 9(l): 79-90. Hildich, T.P., 1940. Chemical Constitution of Natural Fats. Chapman and Hall, London. Hohnan, R.H., 1978. Quantitative chemical taxonomy. Prog. Chem. Fats Lipids, 16: 9-29. Koroleff, F., 1983a. Determination of total silicon. In: K. Grasshoff, K. Ehrhard and K. Kremling @da), Methods of Seawater Analysis. Verlag Chemie, Weinheim, pp. 180-182. Koroleff, F., 1983b. Total silicon by carbonate fusion. In: K Grasshoff, K Ehrhard and K. Kremling (Eds.), Methods of Seawater Analysis. Verlag Chemie, Weinheim, p. 183. Malej, A. and J. Faganeli, 1988. In: 11 fenomeno de1 mare sporco nell’Adriatico (luglio-agosto 1988), Consiglio Nazi* nale alle Ricerche, Roma-Trieste, pp. 47-49. Milani, L., M. Cabrini, S. Fonda Umani and G. Honsell, 1987-1988. The microzooplankton in a station in the Gulf of Trieste from March 1986 to September 1988: data report. Nova Thalassia, 9: 53-95. Pagliarani, A., M. Pirini, G. Trigari and V. Ventrella, 1986. Effect of diets containing different oils on brain fatty acid composition in sea bass (Dicentrarchus labrad. Comp. Biochem. Physiol., 83B: 277-282. Parker, P.L., C. Van Baalen and L. Maurer, 1967. Fatty acids in eleven species of blue-green algae: geochemical signiflcame. Science, 155: 707-708. Parsons, T. and M. Takahashi, 1975. Chemical composition Phytoplankton. In: Biological Oceanographic Process. Pergamon, oxford, pp. 39-49. Pohl, P. and F. Zurheide, 1979. In: H.A. Hoppe, T. Levring and Y. Tanaka (Eds.), Marine Algae in Pharmaceutical Science. Walter de Gruyter, Berlin, pp. 473-523.
R viviani et al. /Science of the Total ‘Environment I65 (1995) 193-201 Rajendran, N., Y. Suwa and Y. Urushigawa, 1992. Microbial community structure in sediments of a polluted bay as determined by phospholipid ester-linked fatty acids. Mar. PoIIut. Bull., 24(6): 305309. Rinaldi, A., G. Montanari, C.E. Ferrari, A. Ghetti and R.A. Vollenweider, 1992. Mucilages in Italian sea: the Adriatic and ‘Qrrhenian sea during 1988-1991. In: Workshop on the Mucilage Phenomenon of the Adriatic Sea and Similar Problems of Elsewhere. Cesenatico, ItaIy, September, 1992. Schleifer, R.H. and Stackebrandt, 1983. Molecular systematics of Prokaryotes. Annu. Rev. Microbial., 37: 143-187.
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Stachowitsch, M., N. Fan&o and M. Richter, 1990. Mucus aggregates in the Adriatic sea: an overview of stages and occurrence. Mar. Ecol., ll(4): 327-350. Strickland, J.D.H. and T.A. Parsons, 1%8. A practical handbook of sea water analysis. Bull. Fish. Res. Board Can., 167. Utermohl, H., 1958. Zur Vervohkommung der quantitativen Phytoplankton, Method&. Mitt. Inter. Ver. Limnol., 9: 1. UNESCO, 1991. IGC-SCOR Workshop on Programme Development for Harmful Algal Blooms. Newport, RI, 2-3 November 1991.
Fatty acids, chlorophylls and total silicon in mucilaginous aggregates collected in a coastal area of the Northern Adriatic Sea facing Emilia-Romagna in August 1988 R. Viviani*“, L. Boni”, 0. Cattania, A. Milandrib, M. Pirini”, R. Polettib, M. Pompeib “Centro
di ricerca interdipartimentale bConsoRio di studi, ricerche
de& scienze dei mare, Uniwrsitli di Bologna, Bologna, e interventi sulle risorse marine di Cesenatico, Forli Italy
Italy
Abstract
Fatty acid, chlorophylls a&c and total silicon analysis were carried out in mucilaginous aggregates collected in a coastal area of the Northern Adriatic Sea facing Emilia-Romagna in August 1988, with the aim of contributing to determine the taxonomy of the producers. The combined data seem to con&m the idea that the mucilaginous aggregates mostly derive from the diatoms responsible for typical blooms in the Adriatic sea. When the presence and morphology
of the organisms
in the mucilaginous
aggregatesis not clear, the biochemicalmarker study seemsto be
useful not only for taxonomic determination of marine micro and macro algae and other eukariotic organisms, but alsofor marine prokariotes. Keywords:
Mucilaginous aggregates; Adriatic Sea; Microalgae; Macroalgae; Diatoms; Dinoflagellates; Phytoplankton
1. Introduction
During July and August 1988, a ‘dirty sea’ phenomenon characterized by very large quantities of mucilaginous aggregates not previously observed in the last 40 years, was recorded along the coasts of the northern and middle Adriatic (Brambati, 1988; De Gobbis, 1989). The first signs occurred at the end of June at Lussino and on 26 July at Rovigno (Bressan,
* Corresponding author. 004%9697/95/$09.50 SSDZ
1988). The collection and studies of mucilaginous aggregates were carried out by the laboratories of Portorose, Trieste, Venice, Cesenatico, Fano and Ancona. As the phenomenon of ‘dirty water’ has been connected with diatoms (since the last century), studies have been directed towards pelagic and benthonic diatoms. At Portorose in the ‘marine snow’ accumulated in the south-east area of the gulf of Trieste, Malej and Faganeli (1988) noted diatoms, the most prevalent being Nitzschiu closterium, Leptocylindrus dunicus, Chaetoceros simplex, and Skeletonema costatum.
0 1995 Elsevier Science BV. All rights resewed.
0048-9697(95)04552-X
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According to Fonda Umani et al. (1988) at Trieste, few animal organisms and phytoplankton cells were evident in the material collected on 13 August 1988, while on 16 August, there were enormous quantities of pelagic diatoms (Nitzschiu, spp., Leptocylindms sp., Chaetoceros spp., and Skeletonemu co.stutum). From 17 August onwards, the same quantities of diatoms were no longer present in the mucilaginous aggregates. In the Venice laboratories, the examination of the material indicated the highest abundance of the diatom Nitzschia closferium (France, 1988). During the monitoring programme of eutrophication along the Emilia Romagna coast in the mucilaginous aggregates collected for the first time on 8 August at Ravenna and examined in the Cesenatico laboratories, it has been demonstrated that the dominant species of diatoms was Nitzxhia clostetium and, among the dinoflagellates, Gonyuular frugilis. On the same day, in material collected at Punta Marina, the dominant diatoms were Skeletonema costatum, Nittschia closterium, and lrhalassiosirasp., while the dominant species of dinoflagellates were Gonyaz& polyedru and Oxytoxum Zongiceps(Consorzio Cesenatico, 1988). At the beginning of August, at the same time as the mucilaginous aggregates appeared, there was a bloom of phytoflagellates probably Chrysophyceae (14 million cells per I) at Port0 Garibaldi. The mucilaginous aggregates collected on 12 August 1988 at Marina Romea had Nitzschia closterium and Thalassiosira sp. as dominant diatoms and for the dinoflagellates Gonyuuluxfiagilis, while those on 16 August collected at Bellaria contained Nitzschia closterium and Gonyaulax frugi1i.s in addition to fragments of macroalgae (Consorzio Cesenatico, 1988). We have attempted a biochemical taxonomic approach with the aim of contributing to the knowledge of algal organisms from which the first mucilaginous aggregates formed, and which subsequently included other micro- and macroalgae (and also some microzooplankton and micrometazoan organisms), and which were subjected to the action of marine bacteria. In this report, we have evaluated whether it could be useful to take into consideration the
pattern of fatty acids of total lipids, chlorophylls a, b, c and total silicon analysis of mucilaginous aggregates in order to ‘finger print’ the material with regard to its origin and to contribute to the knowledge of the molecular nature of mucilaginous aggregates in their tinal stages. Following the suggestion by Hildich (1940) on the role of lipid constituents on quantitative chemical taxonomy, a voluminous literature on fatty acids composition has been generated for use in the description and classification of species (Holman, 1978). Various investigations have revealed that the fatty acid composition of lipids are chemotaxonomic markers useful not only in the classification of micro- and macroalgae (Parker et al., 1967; A&man et al., 1968; Parsons and Takahashi, 1975; Pohl and Zurheide, 1979; Harwood, 1988; Harwood et al., 1988) (Table 11, but also for prokariotes (Cyanobacteria and Eubacteria) (Parsons and Takahashi, 1975; Schleifer and Stackebrandt, 1983; Fulco, 1983; Harwood et al., 1988; Rajendran et al., 1992). Also, chlorophylls u, b, c and total silicon could give additional taxonomic indications for microand macroalgae. Cyanophyceae have chlorophyll a only, Chlorophyceae a and b, Bacillariophyceae (Diatoms) and Dinophyceae (Dinoflagellates) a and c. Silicon is present in the frustule of diatoms as hydrated amorphous silica (SiO,*nH,O), but it is absent in other algae. In this report, fatty acids (Table 2) of total lipids (Table 3) were determined in a sample of mucilaginous aggregate, collected on the sea surface on 16 August 1988, about 2 km off the west coast of Bellaria, at the central point of the Emilia-Romagna coast. In order to have additional taxonomic indications, chlorophylls a, b, c, and total silicon (Table 3) were analyzed on a dry weight basis. To evaluate the chemical and biochemical data obtained from samples of mucilaginous aggregates in relation to the species of phytoplankton present in the sea water column in August and in the preceding periods, data on phytoplankton blooms in the area of the sea off the coast of Emilia-Romagna from the months of June-August 1988 (Table 4) have been reported. Of the blooms found, only the most abundant
17.2 -
9.4 -
0.6 2.6 2.5 -
3.7 -
1.8
0.4 0.4 25.4 24.9
5.5
0.6 2.2
1.1
-
0.4 1.7
-
3.7
0.5 1.2 20.8 -
-
0.5 0.1
3.4 0.5
0.3
2.9 8.7 1.2
0.1
13.8 -
-
-
0.5
11.0 21.2
-
0.6 6.3
0.1
1.4 6.9 22.4 7.4 2.3 6.6
1.0
0.2 32.7
Bacillariophyceae (diatoms, phytoplankton) A B C
-
3.2
6.5 4.3 19.8 -
-
0.2 0.4
0.4 11.3 21.7 2.8 9.4 1.2 0.2 2.9 0.8 -
-
0.5 6.3
D
polyedra
N = Alexandrium
tamarense
1= Prorocentrum
Values are % of total fatty acids; tr, trace. A = Nitzschia closterium B - Skeletonema costatum
2o:o + 2O:l 20:2 20~3 20:4n-6 20~4 n-3 20:5n-3 22:0+ 22:1 22~4 22:s n-3 2216 n-3
l&O l&l 18:2 n-6 18~3 n-6 18:3 n-3 l&4 n-3
I6:4
12:o 14:o 14:l 150 16:0 16:l 16:2 16:3
Fatty acid
0.4 2.2
8.0
0.1
0.5
tr
5.5
16.4 -
-
-
tr tr
2.7
tr
30.1 38.3 4.4 2.7
-
-
C = Chaetoceros septentrionale micans J = Monocrysis lutheri
-
-
0.2 0.2
23.2 44.8 2.8 6.5 0.1 0.3 0.4 0.4 -
0.1 1.0
7.9
0.1
E 2
3 2
23
3
1 18
-
-
5 12 -
-
3 5 5
28 4 2 -
11 1 -
-
0.5
2.4 6.0
6.4
12.4
-
-
-
-
11.6
-
9.8 22.7
13.6
0.4
0.4 13.1
16.3 1.2 -
-
-
0.2 4.0 0.6 0.1
0.1
0.8 0.7
0.2 11.4 0.2 0.2 15.1 25.4 1.9 0.6 0.3 -
Chrysophyceae (phytoplankton)
9.3 1.6 0.4 16.7 24.2 0.1 0.2 0.2 0.5 0.7 2.1 0.6 0.5 2.2 0.4
0.1
17.9 2.1 0.3 1.3 17.8
-
0.2 0.4
tr
2.3 0.7 0.8 2.6
1.4 1.0
20.8 16.9 0.5 0.2 0.4
0.7 9.4 5.6 0.6
12.5 2.6 0.8 2.9 15.7
-
0.8
0.1
Chlorophyceae (macroscopic) L
tr
0.5
0.5 0.4
45.5 -
-
0.8 0.5 1.3
1.0 16.1 1.7 -
-
18.2 3.4
-
1.9 8.1
Rhodophyceae (macroscopic) M
D = Asterionella japonica E = Thalussiosira fluvialis F = Navicula incerta G - Goniuular K = Ulva lactuca L - Enteromorpha intestinalti M = Gracilaria confervoides.
14 -
3 14 -
-
-
1
36
-
-
Dinophyceae (phytoplankton)
Table 1 Fatty acids composition (% of total fatty acids) of some genera and species of marine phytoplankton and marine macroalgae according to Pohl and Zurheide (1979)
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Table 2 Fatty acid composition of total lipids of ‘mucilaginous aggregates” Fatty acid
Weight %
120 14:Oi 14:o 150 16:Oi 16:0 16:l 17:o 17:l l&O l&l n-9 18:2 n-6 l&3 n-6 18:3 n-3 2o:o 2O:l n-9 l&4 n-3 21:o 20~4 n-6 220 221 n-9 20:5 n-3 240 241 n-9 22:5 n-3 22~6 n-3
1.5 f 0.0 0.3 f 0.0 10.0 f 0.9 1.0 f 0.1 0.2 f 0.0 31.4 f 1.0 20.1 & 1.5 0.5 f 0.0 0.2 f 0.0 4.1 f 0.2 13.0 f 0.6 3.4 f 0.3 0.4 f 0.0 0.7 f 0.0 0.3 f 0.0 0.8 f 0.0 0.3 f 0.0 Traces 1.0 f 0.1 0.4 f 0.0 4.2 f 0.4 4.3 f 0.0 0.4 f 0.0 Traces 0.4 f 0.0 1.1 f 0.1
‘Results expressed as the mean of duplicate determinations f S.E.
Table 3 Total lipids, silicon and phytopigments of ‘mucilaginous aggregates’ expressed as dry weight g/loo g Total lipids Total silicon Chlorophyll a Chlorophyll b Chlorophyll c Pheopigments
mg/lOO g
1.100 6.450 3.520 0.437 6.800 2.720
and stored at -20°C until analysis. In addition after filtration, the material used for determination of total silicon has been treated with ultraturrax before analysis. For each sample, dry weight has been determined (105°C). Samples were used for qualitative determination of phytoplankton species and for macroscopic examination. Investigation of bacteria content and the presence of microzooplanktons and micrometazoan organisms was not carried out. This was the subject of research by other authors in the same period in a coastal area of the northern Adriatic Sea facing Emilia-Romagna (Aulicino et al., 1989) and in the Gulf of Trieste (Cabrini et al., 1987-88, 1989; Milani et al., 1987-88; Fonda Umani and Ghirardelli, 1989). 2.2. Algae in sea water and mucilaginous aggregates
have been reported, those with a value greater than 5 million cells per litre. 2. Materials and methods 2.1. Mucilaginous aggregates:samplepreparation
A sample of mucilaginous aggregate was collected on 16 August 1988 on the surface of the northern Adriatic sea, about 2 km off the coast of Bellaria. The samples were collected in a number of 500-ml polyethylene bottles, skimming the gelatinous component. From this material used in sample preparation for phytoplankton studies and determination of total lipids, fatty acids, pigments and dry weight, the water was filtered through a nylon net of about 20-pm mesh size. The residue (for biochemical analysis) was immediately frozen
For algal studies, the sea water and the material were preserved with Lugol’s solution and examined by Uterrnohl’s (1958) method. For taxonomical studies, the sample was examined with a common light microscope. The phytoplankton data in mucilaginous aggregates are only qualitative. In Table 4, quantitative data of phytoplankton blooms from January to August are reported. 2.3. Total lipids determination and fatty acids analyses
Lipids were extracted according to Folch et al. (1957) from the sample of mucilaginous aggregates. The solvent mixture used for extraction contained 0.01% (w/v) BHT as antioxidant. Total lipids (TL) were determined gravimetrically. The saponification of total lipids, the extraction of
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Table 4 Principal blooms registered in 1988, in the period preceding the appearance of the mucilaginous aggregates in the area of sea off the coast of Emilia-Romagna Month
Station
Phytoplankton
Cells/l
January February April May July August
Lido Adrian0 Lido Adrian0 Rimini Lido Adrian0 Bagni di Volano Port0 Garibaldi
Hemiaulus hauckii (diatom) Thalassiosira sp. (diatom) Skeletonemu costatum (diatom) Chaetoceros spp (diatom) Chaetoceros spp (diatom) Chrysophyceae
5658OW 7242000 50 122500 110500ooo 23 341000 14934875
fatty acids (FA) and the methylation using 14% BF, in methanol were carried out as described by Borgatti et al. (1982). Methyl esters of FA were analyzed by gas-liquid chromatography (GLC) and also by chromatography on silicic acid columns impregnated with 25% AgNO, and by catalytic hydrogenation. GLC was done on a Dani (Monza, Italy) 3600 gas-chromatograph, using a glass column (2 m x 4 m) filled with acid-washed Chromosorb W 60-80 mesh and 20% (w/w) diethylene-glycolsuccinate. The carrier gas was nitrogen. The GLC operating conditions and the methods of identification of FA were carried out as reported by Pagliarani et al. (1986). Methyl esters of FA separated by GLC are given as weight percentages of total FA in total lipids (Table 2). 2.4. Chlorophyll and pheopigments Chlorophylls a, b, c and pheopigments
have been extracted from the sample with 90% acetone and estimated spectrophotometrically according to the method of Strickland and Parsons (1968) (Table 3).
2.5. Total silicon
The samples have been analyzed for total silicon according to Koroleff (1983a,b) (Table 3). Since the presence not only of reactive silicate (the dissohed ions of the orthosilic acid) but also of inorganic and organic fractions of diatom frustules (cell wall) is possible in mucilaginous aggregates, the samples were treated with an alkaline persulphate solution to break down these silicon complexes and convert them to reactive silicate.
3. Results and discussion 3.1. Algae in mucilaginous aggregatesand in sea water
Observations under the optical microscope have demonstrated the presence of an extremely heterogeneous amorphous structure in the samples examined, in which the presence of algal cells of diatoms and dinoflagellates, and fragments of macroalgae can be noted. The qualitative study carried out on phytopiankton species showed the presence of Nitzschia clostetium and Gonyaulax fragilis, the first being clearly predominant. From the comparison between the phytoplankton population present in the sea water column with some species of dinoflagellates and diatoms present in mucilaginous aggregates, the following species were not found in the latter: Gonyaulax po(yedra and Thalassiosira sp. The species Nitzschia clostetium and Gonyaulax @gilis were found both in the sea water and the mucilaginous aggregates. From the data in Table 4, it can also be seen that the phytoplankton blooms of January-August were the following: Hemiuulus hauckii, Thalassiosira sp., Skeletonema costatum, Chaetoceros sp. and Chrysophyceae. 3.2. Total lipids
In the dry weight of the ‘material’, total lipids represent 1.1%. These lipids are constituents of cellular membranes of all algal cells and of the lipid body in some diatoms. Traces of lipids of eubacteria are present, possibly also of microzoo plankton and micrometazoan captured from sea water. The superficial total microzooplankton
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R Viuiani et al. /Science of the Total Environment 165 (1995) 193-201
population in August is characterized by population values of some hundreds of individuals per litre and micrometazoan of some 20 individuals per litre (Milani et al., 1987-1988). 3.3. Fatty acid in total lipids in mucilaginous aggregates
From Table 2, it can be seen that for the fatty acids in the total lipids, 14:0 is present at 9.3%, 16:0 at 29.1%, 16:l at 18.7%, 18:0 constitutes 3.8%, 18:l at 12.1% and Cl8 polyunsaturated at 4.6%; 20:4 n-6 constitutes 0.9%, 20:5 n-3 constitutes 4.09% and 22:6 n-3 constitutes 1.1%. Comparing these data with the quantities of fatty acids characteristic of some types of diatoms and of dinoflagellates presented in Table 1, it corresponds to those present in the mucilaginous aggregates examined. The high percentage of 16:0 and 16:1, the presence of 20:4 n-6, the level of 20:5 n-3, and the lack of 22:6 n-3 are characteristic of the fatty acid spectrum of the diatoms (Skeletonemu, Chaetoceros, Nitzschia) rather than that of the dinoflagellates (Gonyaulux sp.) taken into consideration. In these, in fact, the quantity of 16:l is low (l-4%), while 22:6 n-3 is present in relatively high quantities (18-23%). However, the characteristics noted by us can also be common to any species of Chrysophyceae. Since a bloom of an unidentified species of Chrysophyceae was recorded at the beginning of August 1988 in the area of sea between the mouth of the river PO and Ravenna, one cannot exclude the contribution of this species to the formation of the mucilaginous aggregates examined by us. As far as the levels of 18:l (12.1%) are concerned, they are higher than those reported for the same diatoms and dinoflagellates in Table 1; these could be derived from, as far as the algae components are concerned, the numerous macroalgae present in the coastal area of EmiliaRomagna, above all, Chlorophyceae, of the Ulna genus, and Rhodophyceae such as Graciliaria confervoides and Polysiphonia sertularioides of which minuscule fragments were incorporated in the mucilaginous matrix analyzed.
Nevertheless, even the bacterial component, expressed as CFU/lOO ml, which in the same mucilaginous aggregates is enriched with respect to the average values taken in the same zone in the absence of mucilaginous aggregates (Aulicino et al., 1989) could be the source of mono-unsaturated fatty acids, above all 18:1, which are generally present in high percentages in the eubacteria (Rajendran et al., 1992). The low levels of 14:0 iso (0.3%) and 16:0 iso (0.2%) in addition to 15:0 (l.O%), 17:0 (0.5%) and 17:1(0.2%) in the sample indicates that the quantity of Eubacteria present does not represent a significant contribution to the lipid total of the mucilaginous aggregates. In bacteria, the lipids of Archaebacteria are distinguished from those of Eubacteria by the absence of fatty acid glycerol ester lipids and by the presence of isoprenyl glycerol ether lipids (Schleifer and Stackebrandt, 1983; De Rosa and Gambacorta, 1988). Since fatty acids are not constituents of Archaebacteria lipids, the presence of these marine bacteria could not be excluded in the sample analyzed. Concerning eubacterial lipids normal-chain fatty acids, iso and anteiso branched chain fatty acids containing 14-17 carbon atoms are the dominant acyl components of glycerol esters. In effect, with the exception of Archaebacteria, all prokariotes and eukariotes synthesize fatty acids (Fulco, 1983). Since previous blooms of diatoms Skeletonema spp. and Chaetoceros spp. (Table 4) could be responsible for the origin of ‘marine snow’, C16, C18, C20, and C22 polyunsaturated fatty acids could have undergone chemical autoxidation in the presence of oxygen or bacterial metabolic modification, like for example biohydrogenation in an anaerobic environment. In addition, since primitive ‘marine snow’ and the final ‘mucilaginous aggregates’ could capture some microzooplankton and micrometazoan organisms, it is not possible to exclude the presence of their fatty acids. Nevertheless, their presence cannot be proven by particular fatty acids as is possible in the case of bacteria which possess uneven and branched fatty acids.
R Viviani et al. /Science of the Total Environment 165 (1995) 193-201
3.4. Chlorophyll
The content of chlorophylls, expressed in mg/lOO g dry wt., is as follows: chlorophyll a, 3.52 mg; chlorophyll b, 0.437 mg; chlorophyll c, 6.80 mg; pheopigments, 2.27 mg. As the highest levels of chlorophyll are those of a and c, this indicates that the material is derived from diatoms and dinoflagellates. The lower levels of chlorophyll b suggest that it originates from micro and macro Chlorophyceae and, perhaps from a particular Gymnodinium sp. Since Gymnodinium spp. were not present either in the mucilaginous aggregates, or in the previous blooms, the data confirm that chlorophyll b is derived from Chlorophyceae. 3.5. Total silicon
In the dry weight of the samples examined, total silicon represents 6.54%. These high levels of total silicon indicate the presence of diatom frustules, because the concentration of suspended silicon (reactive silicate) is very small in sea water, usually exceeding a few pmol/dm3 (Koroleff, 1983a). Also according to De Gobbis (19891, as appears from the SEM-micrograph of the mucilaginous aggregates collected along the coast of Istria in August 1988, diatom frustules dominated over biogenic remains. The data suggest that in the final stage of mucilaginous aggregates, when large amounts of diatom frustules are present, in addition to the alkaline persulfate method (Koroleff, 1983a), a carbon fusion method could also be utilized (Koroleff, 1983b). 4. Conclusion The data on fatty acids, chlorophylls a, b, c and total silicon combined with the information on the previous and present blooms of phytoplankton and macroalgae could contribute to the knowledge of the evolution of mucilaginous aggregates and their molecular nature in their final stages. These combined data seem to confirm the idea that the mucilaginous aggregates derive from the diatoms responsible for typical blooms in the
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Adriatic Sea, and also to the presence of material derived from Chlorophyceae and Eubacteria. The fatty acid composition of total lipids of samples of mucilaginous aggregates indicates that in the final stage in which there is no demonstrable presence of alga responsible for the phenomenon, the composition of fatty acid in this material can have a taxonomic significance. Our data confirm the findings of other authors (Herndl and Peduzzi, 1988; Stachowitsch et al., 1990; Rinaldi et al., 1992) that planktonic diatoms, such as Skeletonema and Chaetoceros that make up a major fraction of the usual spring bloom of plankton are the main producers of mucilaginous aggregates in the Adriatic Sea rather than dinoflagellates. As far as the condition of phytoplankton in the preceding months of 1988 is concerned, in April, a bloom of Skeletonema costutum was observed with 50 million cells per litre at Rimini, while, in May, all along the coast of Emilia-Romagna a bloom of Chaetoceros spp. appeared which reached 110 million cells per litre of water at Lido Adrian0 (Table 4). Also, high levels of Chaetoceros spp. from Istria to the PO delta (increasingly so near the latter), was reported in May (De Gobbis, 1989). In June, a reduction occurred, while in July at Bagni di Volano, there was a peak reached of 23 million cells per litre. In August, the presence of Nitzschia clostetium was one of the main characteristics of the material collected in the high Adriatic Sea. As the succession of diatoms in bloom in the Northern Adriatic Sea usually starts with Skeletonema and is followed by Chaetoceros and Nitzschia, it will be necessary to evaluate the role of these diatoms at the beginning and in the various phases of the complex phenomenon, which leads from the ‘marine snow’ to the formation of the mucilaginous aggregates. It will also be interesting to evaluate the participation of other algae and of some dinoflagellates of the Gonyuulax genus and the action of marine prokariotes. In all phases of the research, when the morphology of the organisms is not clear, it could be useful, in the area of chemical and biochemical analysis, to examine the spectrum of the fatty
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