ANNALES 11/'97 original scientific paper UDC 593.16(262.3-17) TEMPORAL DISTRIBUTION OF ALEXANDRIUM SPP. IN THE GULF OF TRIESTE (NORTHERN ADRIATIC) Patricija MOZETIČ Marine Biological Station Piran, Si-6330 Piran, FornaCe 41 Marina CA8RINI, Sara ČOK, Riccardo CHIURCOAlfred BERAN laboratory of Marine Biology, 1T-3401G Trieste, str. Costiera 336 ABSTRACT A monitoring program was carried out in the Gulf of Trieste in order to check the quality of shellfish and seawater in which blue mussels (Mytilus galloprovincialis) are cultivated. The occurrence and temporal distribution of dinoflagellate Alexandrium spp., together with environmental conditions in inshore waters, were repotted monthly to biweekly from 1994 to 1996. No blooms occurred, but in May 1994 4200 cells H of Alexandrium species were found along the southeastern side of the Gulf, while on the northwestern side densities up to 4000 cells H peaked in July 1995. An unknown species for the area, presumably related to A. acatenella, was observed for the first time with the scanning electron microscope. Key words: dinoflagellates, Alexandrium spp., environmental factors, PSP toxicity, Guff of Trieste INTRODUCTION The regular occurrence of toxic dinoflagellates is well documented in the Northern Adriatic Sea (Boni, 1983; Honsell et al„ 1992; Mozetič & Obal, 1995). Scientific attention increased particularly since the first DSP (diarrhetic shellfish poisoning) intoxication in 1989 (Boni et al., 1992; Sedmak & Fanuko, 1991) to which Dinophysis (Dinophyceae) species were connected as the potentially causative organisms. Since then, moni­toring programs were set up to identify potentially toxic dinoflagellates and their temporal occurrence as well as toxicity of blue mussels on shellfish farms of the Gulf of Trieste (Northern Adriatic). Up to now no cases of PSP (paralytic shellfish poisoning) human intoxication have been recorded from the Northern Adriatic, although PSP toxins have been detected in blue mussels from the Emilia Romagna (Italy) coast (Honsell et ai, 1996). Since the first obser­vation of the genus Alexandrium on the western side of the Northern Adriatic in 1982 (Boni et a!., 1983), five species of the genus have been identified (Honsell et ai., 1992, 1996). However, there is still a lack of infor­mation regarding their temporal distribution and dyna­mic on a yearly basis. The aim of this work was to study the seasonality of Alexandrium species in the Gulf of Trieste, together with the main environmental factors. MATERIALS AND METHODS The Gulf of Trieste (Fig. 1) is a shallow, semi-en­closed embayrnent with a maximum depth of ca. 25 m in its central part. It is characterized by large tempe­rature variations (6-26°C in the surface layer and 6-20°C above the bottom) and, following the seasonal freshets, surface salinity oscillations (<30-38.5). Along the north­western (Italian) coast there are large mussel farms, while on the southeastern (Slovenian) part there are three shellfish farms. Six stations near the shellfish farms were included in the monitoring program. On the Italian side, the sampling for temperature, salinity and Alexandrium abundance was carried out monthly from July 1994 to December 1996 at stations 225 Slovenia Fig. 1: Sampling locations in the Gulf of Trieste (Adri­atic Sea): (®) stations close to the shellfish farms and (§.0 océanographie stations. Si. I: Vzorčevalna mesta v Tržaškem zaftVo (jadransko morje): (®) postaje v bližini školjčnih nasadov in (H J oceanografske postaje. AO, DO and B. Station B was from May 1995 to De­cember 1996 replaced with station CI . Water samples (500 ml) for cell counts were collected at 0 m, 2 m, 5 m and above the bottom. Stations AO and B are 10 m deep, while the depths of stations C I and DO are 17 and 5 m, respectively. Samples for cell counts (800 ml) were collected bi­weekly at the subsurface (2.5 m) from April 1994 to November 1996 at stations 1 and 2 on the Slovenian side. From January 1994 to December 1996, tempe­rature, salinity, phosphate and inorganic nitrogen (nitra­te+nitrite+ammonium) concentrations were measured at two stations (F and MA) close to shellfish farms 1 and 2 (Fig. 1). Nutrients were measured at 0, 5, 10 m and above the bottom (21 and 16 m at stations F and MA, respectively), although, only mean concentrations from 0 and 5 m were used for this purpose. Temperature and salinity profiles were recorded with a CT D probe, while nutrients were analyzed on un­filtered samples using standard colorimetric procedures (Grasshoff, 1976). Temperature and salinity data were used to calculate "bulk" density gradient (c) as follows c * (0Tb-0Ts)/ H where (7ys and Orb are surface and bottom densities (kg nr3), respectively, and H is water column depth (m). Alexandrium species were identified irs formal­dehyde fixed subsampies and counted on the inverted microscope with a magnification of x200. Some samples with a high abundance of Alexandrium spp. were also prepared for scanning electron microscopy using a SEM Leica Cambridge Stereoscan 43UL Both microscopic methods, optica! and electronic, are described in detail in Zingone ef ai (1990). IKI 3.00 2.00 1.00 S i a g 3 " O .oo 0.80 O " | • MA s S L 0.40 + A® I • Cl [ 99 0.40 A » i 0.20 + gO «'D , & aÎA 0.00 j M M J SNJMMJSN.fMMJ S N MoBibs (1994-1996) Fig. 2: "Bulk" density gradient at six stations in the period 1994-1996. SI. 2: Vertikalni gostotni gradient na šestih postajah v obdobju 1994-1996. RESULTS AN D DISCUSSIO N Hydrography and nutrients During the investigated period the surface tempe­ratures ranged from 7.3°C to 27.2"C. A decrease in sur­face salinity was observed in the periods of higher freshwater discharge, especially in [ate spring and au­tumn (from 27 to 34), while in other months higher salinity values were measured {>34 to 38). The ex­ception was station DO, which is directly influenced by the Isonzo River, the iargest freshwater source in the Gulf of Trieste. At this station extremely high salinity oscillations were observed in the surface layer (from 17 to 37). Stability of the water column is described with a "bulk" density gradient (Fig. 2). Generally, from April to October the water column was stratified (o0.05) , while during the other months it was mixed. Station DO is shown separately on Figure 2, because the water column was most of the time density-stratified due to continuously diluted surface layer. In the period of thevrnohaline stratification, A/exandrium spp. was found in the seawater samples. At the F and MA mean phosphate concentrations in the subsurface layer were most of the time below 0.10 or even 0.05 pmol H (Fig. 3). The highest concen­trations (from >0.20 up to 0.30 pmol I"1) were measured in the summer periods of 1994, 1996, November 1994 and February 1996. Generally, mean phosphate concen­ 226 ANNALES 1 t/'97 Patrie! jn MOZETI Č el ai..-TEMPORAL DISTRIBUTIO N O F AUiXANORIUM SPP. I N TH E G U t F .. , 22S-230 Fig. 3: Mean phosphate and DIN (dissolved inorganic nitrogen) concentrations at stations F and MA in the southeastern part of the Gulf (1994-1996). SI. 3: Povprečne koncentracije fosfata in raztopljenega anorganskega dušika (DIN) na postajah F in MA v jugo­vzhodnem delu zaliva (1994-1996), traiions increased from May to November in the years 1994 and 1996, whereas in 1995 the highest concen­trations {>0.05 pmol H ) were measured in the first three months and July. in the upper water column, nitrate was the predo­minant form of dissolved inorganic nitrogen. Highest mean concentrations (around 10 pmoi H ) were found in the winter-spring and autumn periods (January-March, November), and only once in the summer period (July 1995) (Fig. 3). The peaks of inorganic nitrogen, espe­cially nitrate, are connected to externa! nitrogen inputs from land during spring and autumn freshets of the Isonzo River, and occasionally with rain during the summer storms (Malej et a!., 1997). During late spring and summer, concentrations of dissolved nitrogen de­creased and became the lowest in July 1996 (1.0 po l H) . Besides low phosphate concentrations, calculated high N/P ratios (on average 58) reflect limited phosphate availability during almost the whole investigated period. Only during iate spring-early summer in 1994 and 1996 the N/P was close to or below the Redfield ratio of 16. Temporal and spatial dynamics of Alexandrium species As the identification of Alexandrium species is very difficult with optical microscopy, we classified all the observed species as Alexandrium spp, Alexandrium species were present in the samples during almost each of the months of the 1994-1996 period, but their abundance increased from April to October each year (Fig. 4). !n this period cell numbers ranged from unde­tectable to 4000 and 4200 cells H in the northwestern and southeastern part, respectively. O n both sides of the Gulf maximal densities were quite similar, but the months of peak abundances were different. The highest abundance was recorded in May-june in the south­eastern part, while in the northwestern part abundance peaked later in the season (July-August). Following these peaks, abundance decreased significantly to increase slightly again in September and/or October at some sta­tions. In the northwestern part cells counted at discrete depths were integrated in two layers: subsurface, low-salinity iayer (from 0 to 5 m), and a deeper layer above the bottom. Generally, higher abundance was found in the subsurface layer, whereas at station B maximal densities were at 10 m. Among Alexandrium species we identified A. pseu­dogonyaulax and A. minutum, two species already reported from the Gulf of Trieste (Honsell et ai, 1992). During the abundance maximum of genus Alexandrium in May 1994 new species for this area was found in the southeastern part. Based on scanning electronic obser­vations it is presumably related to A. acatenella (Fig. 5; Fukuyo, pers. comm.). Interestingly, in 1996 a similar species appeared in the samples from the northwestern part and it was isolated. Although more detailed mor­phological study has to be done to ascertain the taxo­nomic position of A. cf. acatenella, it is likely that a new Alexandrium species did appear on both sides of the Gulf of Trieste. In our case the isolated species is now kept in cultures for morphological and possibly bio­chemical (HPLC analyses for PSP toxins) studies. Environmental factors and Alexandrium abundance The seasonality of Alexandrium spp. in the Gulf of Trieste is similar to those observed in other Mediterra­nean areas, with even higher maximal densities espe­cially in the upper, less-saline layer and in connection with developed thermohaline stratification (Delgado et ai, 1990; Gtacobbe et al., 1996). In our study we ob­served that the increased cell numbers coincided with the decreased surface salinity in late spring-early sum­mer at all stations. The exception was again station DO, where in a very diluted surface iayer (salinity <20, April 1995) Alexandrium spp. was not found, but was present at 2 m (salinity >30), Although intense autumn freshets caused another surface-salinity decline, Alexandrium abundance did not increase. This is probably linked to the destratification of the water column and lower temperatures compared to the spring-summer period. Higher temperatures, a stratified water column and ab­ 227 Patricijil MOZETI Č et al. .-TEMPORAL DISTRIBUTIO N O F ALBCANOHIUM S PP. tN TH E GUI F ..., 225-230 4000 2000­ Want Sfl 4000­JMMJSNJMMJSNJMMJSN » 2000. Jr. 1a S « H s»•!S 400­300­200­100­0.. J 300­, UJUJMMJSNJMM J S N J M M j S N 250. Cl 200 150 100 50­| 0 . , . l-^jfl-T-v, -P-1ÇI JMMJSNJMMJSNJMMJSN Months (1994 -1996) 2000 subsurface above bottom 1500 1000 500 JL *r JMMJSNJMMJSNJMMJSN 4000 2000 400 300 200 100. JMMJSNJMMJ SNJMMJ S N 4000 2000 400­300 200. 100­0 ÎU4U1 M JMMJSNJMMJSNJMMJSN Months (1994 -1996) Fig. 4: Aiexandrium spp. abundance at six stations in the period 1994-1996. (Note the different units on y axes.) SI. 4: Gostota vrst iz rodu Aiexandrium na šestib postajah v obdobju 1994-1996. (Upoštevaj različne enote na oseh y-) sence of turbulence seem to be favorable conditions not only for Aiexandrium but for di no flagellates as a group (Paerl, 1988). in mixed and nutrient enriched autumnal conditions diatoms prevail in the phytopiankton com­munity of the Gulf of Trieste (Malej et al., 1995; Cabrini et al., 1994) As mentioned above, mean phosphate concentra­tions increased from May to November in the years 1994 and 1996, and in }uly 1995. These periods coin­cide with the occurrence of Aiexandrium species at the nearby stations 1 and 2. However, during the periods of favorable phosphate conditions Aiexandrium abun­dance peaked (>1000 cells H ) only on few occasions (May 1994, 1995, 1996, June 1996), suggesting that 228 ANNALES 11/'97 Pa tri ci ja MOZETIČ ei 3Î.:TEMPORAL DISTRIBUTION OF AOXANORIUMS PP. I N TH E GU1.F ..., 22S-2ÎQ factors other than phosphate availability control the Alexandrium dynamics. Coincidence of high tempe­rature, low salinity, stability of water column, high phosphate concentration and low N/P ratio, with high density of Aiexandrium spp., was also observed in the Sicily lagoon, Mediterranean Sea (Giacobbe et a/., 1996). Several studies have also shown strong relation­ship between phosphate limitation and toxin production in Aiexandrium species {Boyer ei al1987; Anderson ef ai, 1990). However, in our case the results should be interpreted with care for at least two important reasons. Aiexandrium abundance and nutrient concentrations were not measured in the same water sample, but from two, although close, different locations. It means that nutrient data allow to give an idea of the general situation in the area. Secondly, an important ecophysiological characteristic that should be taken into consideration is the ability of many free-living dino­fiageliates to perform diel vertical migrations through the water column (Eppley & Harrison, 1975). For example, in our case inorganic nitrogen concentrations were low during the period of Aiexandrium occurrence, which might suggest that Aiexandrium cells could utilize deep nitrogen sources through vertical migrations. This behavioral adaptation enables a population of toxic dinoflageilates to persist in many nitrogen-depleted, summer-stratified surface coastal waters (Koizumi et a!., 1996; Maclntyre et ai, 1997). in conclusion, our study confirmed the presence of Aiexandrium spp. in the Gulf of Trieste and its increased abundance from April to October. Maximal cell num­bers were recorded during thermobaline stratification of the water column, in a less-saline subsurface layer. A more detailed study on the influence of other envi­ronmental factors, mainly nutrients, on Aiexandrium seasonality in the Gulf of Trieste is to be carried out in the future. Fig. 5: Scanning electron micrograph of Aiexandrium sp. (presumably related to A. acatenella) from the sout­heastern part of the Gulf of Trieste. SI. 5: Posnetek (elektronski mikroskop) vrste Aiexan­drium sp. iz jugovzhodnega dela Tržaškega zaliva, ki je najverjetneje sorodna vrsti A. acatenella. ČASOVN A PORAZDELITE V VRS T IZ ROD U ALEXANDRtUM V TRŽAŠKE M ZALIV U (SEVERNI JADRAN) Patricija MOZETIČ Morska biološka postaja Piran, Si-6330 Piran, Fornače 41 Marina CABRINI, Sara ČOK, Riccardo CH1URCO, Alfred BERAN Laboratorio cii Biologia Marina, iT-34010 Trieste, str. Costiera 336 POVZETEK V prispevku avtorji podajajo rezultate triletnega spremljanja gostote toksičnih vrst dinoflagelatov na šestih po­stajah v bližini školjčnih nasadov užitne klapavice vzdolž italijanske in slovenske obale Tržaškega zaliva. Dino­flagelati iz rodu Alexandrium so povzročitelji paralitične zastrupitve s školjkami (PSP) pri ljudeh, zato je že nekaj let na obeh straneh zaliva vpeljan program rednega spremljanja kakovosti morske vode in školjk. Poleg časovne in prostorske porazdelitve celic Alexandrium spp. v obdobju 1994-1996 so avtorji spremljali tudi nekatere fizikalno­kemijske parametre (temperatura, slanost, hranilne snovi). Vzorčevanje v mesečnih do dvotedenskih presledkih od leta 1994 do 1996 je pokazalo, da se Aiexandrium spp. pojavlja skoraj v vseh mesecih, vendar njegova gostota naraste v obdobju od aprila do oktobra vsako leto. V jugo­vzhodnem delu zaliva je bila največja gostota zabeležena v maju 1994 (4200 cel. H), v severozahodnem delu pa julija 1995 (4000 cel. h1). Ti spomiadansko-poletni viški so se ujemali z gostotno razslojenostjo vodnega stolpca, 229 ANNALES 11/'97 Pairtdja MOZETI Č ei ai..-TEMPORAL DISTRIBUTIO N O F ALEXANDKIUM SPP IN TH E GUL F ..., 225-230 kot posledico povišanih temperatur in oslajevanja zgornjih slojev. V obdobju naraščajoče gostote vrst iz rodu Alexandrium so bile v jugovzhodnem delu zaliva izmerjene povečane koncentracije fosfata, vendar so bili viški števila celic (>1000 cel. H) izmerjeni le štirikrat v daljšem obdobju ugodnejših fosfatnih razmer. Avtorji opozarjajo na previdnost pri razlagi sezonske dinamike roda A!exandrium zlasti zaradi dveh pomembnih dejstev: 1. število celic in koncentracija hranilnih snovi so bili merjeni na dveh različnih, četudi blizu ležečih po­stajah, zatorej izmerjene koncentracije hranilnih snovi odsevajo zgolj splošne hranilne razmere na tem območju; 2. za dinoflagelate je značilna dnevna vertikalna migracija po vodnem stolpcu, kar jim omogoča izkoriščanje globljih, bogatejših virov hranil zlasti v plitvih, temperaturno razslojenih priobalnih vodah. V vzorcih morske vode je bila z elektronskim mikroskopom odkrita do sedaj nepoznana vrsta Aiexandrium sp. iz Tržaškega zaliva, ki je najverjetneje sorodna vrsti A. acatenelia. Vendar so za pravilno taksonomsko določitev in morebitno toksičnost potrebne nadaljnje morfološke in biokemijske raziskave na monokulturi izolirane vrste. 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