There are several small karstic rivers that discharge into the eastern Adriatic Sea, forming highly stratified estuaries that have not been adequately investigated. High stratification is maintained in areas where a high volume of river discharge is combined with low tides (Dyer 1991). Estuaries are highly productive habitats, providing ecologically and economically valuable fish-larvae and shellfish refugia, nurseries (Steele 1974), and dynamic nutrient transformation zones at the interface between freshwater and marine environments (Nixon 1995). Temporal and spatial distribution of phytoplankton in an estuary is regulated by the dilution of river flow, light and mixing processes (Cloern 1996). The increased primary production in estuaries is influenced by nutrients brought by the river discharge (Malone et al. 1988).

Highly stratified estuaries are characterized by an accumulation of microphytoplankton (ViliČiĆ et al. 1989), nanophytoplankton (Denant et al. 1991, Ahel et al. 1996), bacteria (Fuks et al. 1991), dissolved organic matter and detritic particles (ŽutiĆ and LegoviĆ 1987, Cauwet 1991), and pollutants (Mikac et al. 1989) along the sharp halocline.

The scope of this paper is to present a fragment of the interdisciplinary research in the Zrmanja Estuary that was carried out extensively in both 1981/1982 and 1998/1999. We present the seasonal distribution of thermohaline characteristics, phytoplankton and microzooplankton in the middle reach of the Zrmanja Estuary. The results may give some information valuable for further ecological research.

The Zrmanja River is a small karstic river that discharges into the eastern Adriatic Sea. It is 69 km long, from its source in the Dinaric karst region to the mouth in the Velebit Channel. The estuary and the adjacent coastal sea are westerly oriented between the Velebit Mountain ridge on the north, and large North-Dalmatian plateau to the south and east (FriganoviĆ 1961). The 14 km long upper reach (Fig. 1) extends from the Jankovića buk waterfalls to the wider portion of the estuary (Novigradsko more). The steep banks of the upper portion of the estuary are strongly eroded, making the estuarine bed relatively shallow (mostly about 5 m deep).The middle reach consists of Novigradsko and the southeasterly extension of Karinsko more. The lower reach consists of the narrow strait (Novsko ždrilo), as a conection between Novigradsko more and the Velebit Channel. The lower reach is up to 40 m deep. In Novsko ždrilo strait there is a 19-m deep sill. The influence of this sill on overall circulation in the estuary is not defined to this day.

The tides in the area are rather weak: M2 amplitudes are below 10 cm, and K1 amplitudes are close to 13 cm (e. g. KasumoviĆ 1960).

The average outflow (calculated for the period 1953 -1990) equals 38 m3/s, but may be as high as 456 m3/s (December 1959) and as low as 0.09 m3/s (June 1986). There is a complex water circulation in the karst system in the Zrmanja River catchment area (Bonacci 1999). The numerous permanent and temporary springs along the river are connected with swallow holes ("ponors") in the hinterland. Underwater springs ("vruljas") in the estuary discharge water during rainy (October-December) and snow melting periods (March- May).

The surrounding area is without considerable anthropogenic influence, resulting in moderate phytoplankton abundance (indicating natural eutrophication; ViliČiĆ 1989), low chlorophyll a and nutrient concentrations, as well as a well-oxygenated water column (Tab. 1).

Water samples for the analyses of phytoplankton were collected at station N1 (Fig. 1), at monthly intervals, in the period December 1981 to November 1982, and June 1998 to June 1999. The water column at Station N1 is 20 m deep. Phytoplankton was sampled using 5-liter Niskin bottles, mostly at 0, 2, 4 and 10 m depths. Samples were preserved in a 2 per cent (final concentration) neutralized formaldehyde solution. The cell counts were obtained by the inverted microscope method (Utermöhl 1958). Subsamples of 50 ml were analyzed microscopically, after a sedimentation time of 24 h, within one month after the cruise. Cells longer than 20 <$Q410C0000001140696D3FFEB62709C80443330D828DCE62100904F134E5BAECA4F8>m were designated as microphytoplankton, cells 2-20 <$Q410C0000001140696D3FFEB62709C80443330D828DCE62100904F134E5BAECA4F8>m long as nanoplankton. Cells were counted at a magnification of 400 <$QF80C0000001140696D3FFEB6270CE81941C30D828DCE62100904F134E5ABECA4F8> (1 transect) and 200 <$QF80C0000001140696D3FFEB6270CE81941C30D828DCE62100904F134E5ABECA4F8> (transects along the rest of the counting chamber base plate). Recognizable nanoplankton cells were counted in 20 randomly selected fields of vision along the counting chamber base plate, at a magnification of 400 <$QF80C0000001140696D3FFEB6270CE81941C30D828DCE62100904F134E5ABECA4F8>. The precision of the counting method is <$Q0D0D0000001140696D3FFEB6270CE81641DB0D828DCE62100904F134E5ABECA4F8>10%.

Samples for the analysis of microzooplankton were collected using 5-liter Niskin bottle samplers, fixed in 2.5 per cent neutralized formaldehyde and allowed to settle twice for 24 hours in plastic containers and glass cylinders, reducing the original volume of 5 l to a few milliliters. Counts were done at a magnification of 100 <$QF80C0000001140696D3FFEB6270CE81941C30D828DCE62100904F134E5ABECA4F8> (water immersion objective) using a self-designed chamber (75 <$QF80C0000001140696D3FFEB6270CE81941C30D828DCE62100904F134E5ABECA4F8> 45 mm, bottom glass 1 mm thick) (KrŠiniĆ 1980).

Salinity was determined using a Beckman RS5-3 salinometer, and by argentometric titration. Temperature was measured by Beckman sond and a reversing thermometer.

Rainfall and river water discharge determined thermohaline characteristics in the estuary. Higher river discharge (Fig. 2) induced the formation of an about 4 m thick surface brackish layer (salinity 3.46-30 PSU) at Station N1 in January, May and November 1982 (Fig. 3). The salinity in the marine layer (below the halocline) ranged from 30 to 37.94 PSU. During the strong stratification, a vertical salinity gradient of up to 21.77 PSU was registered in the 2-4 m layer (= 10.88 PSU m-1). A similar hydrological regime was evident during 1998/1999. Similar termohaline conditions were detected by CTD multisond during current research in 2000.

Thermic stratification in summer provided temperatures 22-26 #C above 8 m depth, and a thermic gradient of 5 #C in the 4-10 m layer (= 0.83 #C m-1). Inverted stratification was detected in winter (December-March), with temperatures below than 8 #C in the 0 - 5 m layer. A short appearance of surface ice was detected between samplings in January 1999. Isothermic conditions were determined in April and October.

Secchi disc transparency varied between 5.5 and 7.8 m (Tab. 1).

Increased abundances of microphytoplankton (values higher than 106 cells l-1) were detected in winter-spring, with maximum of 1.8 <$QF80C0000001140696D3FFEB6270CE81941C30D828DCE62100904F134E5ABECA4F8> 106 cells l-1 in April 1999 (Fig. 4). Freshwater phytoplankton did not provide more abundant population above the halocline at Station N1. Instead of freshwater species, the still unidentified, small-celled centric diatom Thalassiosira sp. provided dense population in the brackish layer (above the halocline) in January - May 1982 and 1999.

Marine diatoms and dinoflagellates provided the dominant phytoplankton groups throughout the year. During the intensive development of phytoplankton in spring, diatoms were most important group, as indicated by the ratio between abundance of diatoms and dinoflagellates (Fig. 5). Dinoflagellates dominated in winter and summer.

Seasonal variations of phytoplankton taxa provided evidence of relatively recurrent assemblages in the estuary (Tab. 2). There were several diatoms with maximum abundance in winter-spring period, such as: Pseudo-nitzschia spp., Rhizosolenia stolterfothii, Thalassiosira (brackish, unidentified species), and Bacteriastrum delicatulum. The diatom Leptocilindrus danicus was abundant during summer. Among abundant dinoflagellates, several naked and thecate nanoplanktonic species (10-20 <$Q410C0000001140696D3FFEB62709C80443330D828DCE62100904F134E5BAECA4F8>m size fraction) appeared in summer. Among larger prymnesiophites, an abundant population of Syracosphaera was registered in summer-autumn.

Due to the taxonomic composition, microphytoplankton was composed of 72 diatoms (26 pennatae, 46 centric diatoms), 43 dinoflagellates, 7 prymnesiophytes, 3 chrysophyceae, and 1 euglenophyte (Tab. 3). The following diatoms provided maximum abundance greater than 5 <$QF80C0000001140696D3FFEB6270CE81941C30D828DCE62100904F134E5ABECA4F8> 106 cells l-1: Chaetoceros compressus, Ch. tortissimus, Pseudo- nitzschia sp., Thalassiosira sp., while the most abundant dinoflagellates were gymnodinoid cells and Prorocentrum micans.

Discussion

Due to the rather weak tides in the area, the Zrmanja River may be expected to create a highly stratified estuary, at least during episodes of strong river outflow. The measurements performed in the Zrmanja Estuary indicated that the river influenced the stratification and dynamics not only in the middle reach of the estuary (Station N1) but in the Velebit Channel as well (ViliČiĆ et al. 1999).

Vertical temperature profiles are also of interest (Fig. 3). The thermocline was mostly observed close to the halocline. A subsurface temperature maximum was detected in October 1998 and February 1999. It could be interpreted in terms of a combined effect of radiative heating and reduced heat exchange at the halocline level. Moreover, the accumulation of suspended matter close to the halocline and selective absorption of solar radiation might contribute to its occurrence. The same phenomena have previously been observed in some other east Adriatic estuaries - the Krka Estuary (LegoviĆ et al. 1991; OrliĆ et al. 1991) and the Ombla Estuary (ViliČiĆ et al. 1995).

Freshwater phytoplankton species in the Zrmanja Estuary were scarce, in contrast to the estuary of the River Krka, where freshwater phytoplankton develops in the relatively large freshwater accumulation in front of the estuary (ViliČiĆ et al. 1989). The freshwater phytoplankton, mostly composed of chrysophyte Dinobryon and numerous freshwater, pennate diatoms, sink to the halocline and die due to the osmotic shock, resulting in a decrease of cell density seaward.

The fact that marine phytoplankton accumulates just below the halocline is due to the more favorable nutritive and light conditions, as well as the more stable conditions below the outflowing surface brackish layer.

Marine phytoplankton below the halocline is attractive food for herbivores in the lower reach of the estuary. According to Ruiz et al. (1998) microzooplankton grazing is more responsible for phytoplankton distribution along the salinity gradient in the outer than in inner zone of estuaries. Among heterotrophic plankton in the pelagic community, small copepods and larval stages of copepods (nauplii) accumulated in and below the halocline at N1. Seasonal variations of nauplii, copepodites and small adult copepods run parallel, with maximum development in winter (October-Februray)(Fig. 6). Naked ciliates (10 - 30 <$Q410C0000001140696D3FFEB62709C80443330D828DCE62100904F134E5BAECA4F8>m size fraction) provided two maxima; one in winter, and another in summer. Maximum abundance of ciliates was observed below the halocline in summer, and above the halocline in winter (Fig. 7).

Nanoplankton accumulation around the halocline is due to the accumulation and degradation of organic matter (ŽutiĆ and LegoviĆ 1987), and physico-chemical transformations of organic matter (Eisma et al. 1991). At Station N1 nanoplanktonic dinoflagellates accumulated around the halocline (Fig. 8). These cells 2-10 <$Q410C0000001140696D3FFEB62709C80443330D828DCE62100904F134E5BAECA4F8>m in size probably belong to mixotrophs, participating in the processes of microbial transformation and degradation of organic matter. In summer, ciliates play an important role in the degradation of autochthonous organic matter below the halocline (Fig 7). On the other hand, in winter, during the increased river discharge, these microorganisms probably participated in regeneration of allochthonous organic matter, above the halocline.

Low concentrations of orthophosphates and nitrates (ViliČiĆ and Stojanoski 1987, ViliČiĆ et al. 1999, Tab 1), a phytoplankton abundance with the most frequent values between 105 and 106 cells l-1 (without any extremely high values), as well as an oxygenated and transparent water column, indicate a moderate eutrophication of the Zrmanja Estuary (ViliČiĆ 1989). Data on seasonal variations of nutrient concentrations are still not available. According to some personal observations, sporadic phytoplankton blooms in April usually induce short macroaggregate formations in the research area.

In the middle reach of the Zrmanja Estuary, the halocline divided two phytoplankton communities. Freshwater phytoplankton is transported to the estuary by the riverine water from the small freshwater accumulation in front of the estuary. As compared to data in the upper reach of the estuary, freshwater phytoplankton above the halocline is less abundant at Station N1. This might be due to the sinking of freshwater phytoplankton and decaying of cells along the halocline in the upper reach of the estuary, similarly as in the Krka Estuary (VILIČIĆ et al. 1989, LegoviĆ et al. 1996). Instead of freshwater phytoplankton above the halocline at N1, the abundant brackish diatom Thalassiosira sp. was detected. Marine phytoplankton accumulated below the halocline.

Research should be continued in the direction of defining the budget of nutrients and allochthonous organic matter, current system, limiting growth factors, as well as the seasonal production and regeneration processes in the estuary.

We are indebted to Dr Dušan Trninić of the State Hydrometeorological Institute for supplying us with Zrmanja River discharge data. This research was supported by the Ministry of Science and Technology of the Republic of Croatia, under project 119121.