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Assessment of nutrient limitation in Rijeka Bay, NE Adriatic Sea,using miniaturized bioassay

Darija Vukić Lušić a, Vesna Peršić b, Janja Horvatić b,⁎, Damir Viličić c,Luka Traven a, Tamara Đakovac d, Vladimir Mićović a

a Teaching Institute of Public Health, Department of Ecology, Krešimirova 52 a, 51 000 Rijeka, Croatiab Department of Biology, Josip Juraj Strossmayer University, Gajev trg 6, 31 000 Osijek, Croatia

c Division of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10 000 Zagreb, Croatiad Ruđer Bošković Institute, Center for Marine Research, G. Paliaga 5, 52210 Rovinj, Croatia

Received 22 January 2008; accepted 23 January 2008

Abstract

In this paper we consider the trophic state and ambient nutrient limitation by the trophic index (TRIX) calculation and nutrient enrichmentbioassay using the diatom Phaeodactylum tricornutum at two differently eutrophicated NE coastal Adriatic locations (in the Rijeka Bay area). Thefirst station in Rijeka Port had significantly higher concentrations of dissolved inorganic nutrients and TRIX indices (up to 7.53) than the stationnear Opatija. The DIN/TP ratios in Rijeka Port were lower (11.03 to 44.47) than those in Opatija (38.67 to 82.00), indicating P limitation inOpatija. The nutrient enrichment bioassays revealed P as the key limiting nutrient for the diatom growth at both sites. In addition, silica limitationwas found in all water samples with the exception of the surface water samples in Rijeka Port. Nitrogen limitation was rarely evident.© 2008 Elsevier B.V. All rights reserved.

Keywords: Adriatic Sea; Bioassay; Chlorophyll-a; DIN:TP ratio; Limiting nutrients; Trophic index

1. Introduction

During the last decades, coastal zones have shown increasingevidence of degradation, primarily due to human activities(Marcus, 2004). Although many factors influence coastalprimary production, the nutrient supply is fundamental (Smith,1984; Hecky and Kilham, 1988; Pickney et al., 2001).Mediterranean surface waters are classified as among the poorestin nutrients (oligotrophic) of the world's seas (Turley, 1999;Olsen et al., 2001). However, in deep waters of the western basinthe concentrations of phosphate and nitrate reached in 19940.4 μM and 8.7 μM, respectively (Béthoux et al., 1998).Nevertheless, eutrophication in the Mediterranean appears to beevident in coastal areas. Severe eutrophication is evident inenclosed bays which receive elevated nutrient loads from rivers.or poorly treated domestic and industrial wastewaters (EEA,1999c). An identification of limiting nutrients is necessary for the

selection of appropriate nutrient control measures to mitigateeutrophication effects (Magnien et al., 1992). Therefore, it isimportant to develop robust and effective tools to provideidentification of the most limiting nutrient and techniques forassessing eutrophication (Georgios et al., 2006). Chemicalanalyses have long been used to evaluate the quality of water.The N/P ratio is used to estimate the relative importance ofnitrogen (N) vs. phosphorus (P) as the most frequent limitingnutrients by comparing the N/P ratio to the Redfield Ratio (16,atomic). This approach has been questioned, because N and Pconcentrations are not necessarily proportional to their avail-ability, but still, it gives some rough information about nutrientcontrol in the ecosystem (Wasmund et al., 2001). In laboratoryconditions, nutrient limitation can be quantified by evaluating thegrowth response of algae after nutrient additions. The addition ofa limiting nutrient causes an immediate growth response of testedalgae proportional to the intensity of limitation of that particularnutrient. Therefore, nutrient enrichment bioassays are usefulindicators that show which nutrient has the potential or is mostlikely to limit algal growth (Holmboe et al., 1999; Ault et al.,

Journal of Experimental Marine Biology and Ecology 358 (2008) 46–56www.elsevier.com/locate/jembe

⁎ Corresponding author. Tel.: +385 31 232 638; fax: +385 31 232 642.E-mail address: jhorvati@ffos.hr (J. Horvatić).

0022-0981/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.jembe.2008.01.012

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2000). The quantification of the degree of algal growth responseis of great value for measuring nutrient limitation (Downinget al., 1999).

Our goal in this study was to assess nutrient limitation bydetermining nutrient conditions in situ and to compare themwith a miniaturized bioassay method applied in the laboratory.We planed to determine nutrient limitation at different depths inthe Rijeka Bay (Adriatic Sea), and trophic state under differentanthropogenic influences.

2. Materials and methods

2.1. Study area

The Adriatic Sea is the northernmost part of the Mediterra-nean Sea, and Rijeka Bay is the NE extension of the AdriaticSea with an area of 450 km2. The coastline of Rijeka Bay is115 km long with an average depth of 60 m. In contrast to theNWAdriatic, which receives large amounts of freshwater fromthe Po River (Raicich, 1996), the NE Adriatic is mostlyinfluenced by the oligotrophic, north-westerly, incoming East-ern Adriatic Current from the southern Adriatic and the IonianSea (Gačić et al., 2001). Rijeka Port is more influenced bysewage waters than the tourist resort Opatija.

2.2. Sampling

Monthly sampling was carried out from February 2005 toJanuary 2006 at two stations in Rijeka Bay (Fig. 1): in RijekaPort and near Opatija. The first station is in Rijeka Port (45°19VN, 14°25VE), the second one is near Opatija (45°19VN, 14°18VE). Samples were collected using a 5-liter Niskin bottle at threedepths; near the surface (0.1 m below surface), at the Secchivisibility depth and near the bottom. In Rijeka Port, Secchi depthsamples were collected between 5 and 10 m (mean 6.8 m), whilenear the bottom samples were collected between 10 and 25 m(mean 13.6 m). In Opatija, Secchi depth samples were collectedbetween 7 and 14m (mean 9.6 m), while near the bottom sampleswere collected between 9 and 30 m (mean 18.3 m).

2.3. Analytical measurements

Water temperature (WT) and Secchi visibility (Sec) weremeasured in the field, while conductivity (EC), pH, salinity (S),dissolved oxygen (DO), biochemical oxygen demand (BOD),nutrient concentrations such as ammonium (NH4), nitrite(NO2), nitrate (NO3), total phosphorus (TP) and orthopho-sphates (PO4) were measured in the laboratory by standardoceanographic methods (Parsons et al., 1984). Phytoplankton

Fig. 1. Map showing sampling locations in Rijeka Bay (simplified and modified after Benac et al., 2004).

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chlorophyll-a concentrations (Chl-a) were determined fluor-imetrically (TurnerDesigns TD-700) (Parsons et al., 1984).

The trophic index (TRIX) based on chlorophyll-a, oxygensaturation, total nitrogen and total phosphorus has been proposedfor the characterisation of the trophic state of coastal marinewaters (Vollenweider et al., 1998). This index is defined by linearcombinations of logarithms of four state variables according toGiovanardi and Vollenweider (2004), where nutrients arerepresented by dissolved inorganic nitrogen (calculated as asum of ammonia, nitrite and nitrate; DIN=NH4+NO2+NO3, asμg l−1) and total phosphorus (TP) in μg l−1, chlorophyll-a(μg l−1) as phytoplankton biomass, and oxygen as absolutepercent deviation from saturation (aD%O) in the productive layerindicating the production intensity of the system:

TRIX ¼ Log10 ChlQa� aDkO� DIN� TP½ � þ 1:5ð Þ=1:2: ð1ÞNumerically, the index is scaled from 0 (oligotrophy) to 10

(hypertrophy), and for a water body in a good trophic state, itshould not exceed 5 TRIX units. The scale is divided into fourclasses: 2bTRIXb4, High; 4≤TRIXb5, Good; 5≤TRIXb6,Moderate; 6≤TRIXb8, Poor.

The efficiency coefficient (Eff. Coeff.) was defined as the logof the ratio between the two aggregated main components ofTRIX, biotic (Chl-a and aD%O — factors that are a directexpression of productivity) and abiotic (DIN and TP —nutritional factors), Giovanardi and Vollenweider (2004):

Eff :Coeff : ¼Log10 ChlQa� aDkO½ �= DIN� TP½ �ð Þ: ð2Þ

Low values would indicate low, high values high nutrientutilisation.

2.4. Miniaturized bioassay method

Water samples from Rijeka Bay were evaluated using theunicellular marine diatom culture of Phaeodactylum tricornutumBohlin 1897 strain CCAP 1052/1A, obtained by the CultureCollection of Algae and Protozoa, UK by the laboratoryminiaturized growth bioassay method (Horvatić et al., 2006;Horvatić and Persić, 2007). The algae were cultivated in theculture medium according to ISO 10253 (1995), at 18–20 °C,under 16/8 photoperiod exposed to white light irradiance by PAR100 μmol m−2 s−1. The algal inoculum solution was obtainedfrom a preculture, which was incubated under cultivationconditions and used when cells were exponentially growing (3–5 days old culture). Due to the prior uptake and possible storage ofnutrients, it was necessary to starve P. tricornutum cells beforeexperimental use. Before inoculation the algal cells were washedout from the mediumwith sterile synthetic seawater in which theywere subcultured for 3–5 days. Diatoms are the most abundantphotosynthetic organisms in marine waters. P. tricornutum is oneof the most used algae in the marine bioassays because of its easycultivation and it is also a prospective alga in aquaculture.

Water samples were filtered through the glass-fiber What-man GF/C filter (pore size 1.2 μm) to eliminate particles andstored at −20 °C. After melting, bioassay experiments were

carried out in polystyrene 96-well microplates (TPP, Switzer-land). Bioassay experiment was conducted with six replicates ofcontrols (unenriched water sample diluted with synthetic seawater, 1:1) and six replicates of enriched water samples (watersamples with added N, P, Si and their combinations). Nutrienttreatments were added to water samples singly: nitrogen (N) assodium nitrate (NaNO3) in a final concentration of 150 μM N,phosphorus (P) as potassium phosphate (K3PO4) in a finalconcentration of 20 and 6 μM P and silicate (Si) as sodiumsilicate (Na2SiO3×5H2O) in a final concentration of 50 and12.5 μM Si. Also, three different combinations of thesenutrients were added: NP (150 μM N and 6 μM P), NPSiwith high Si (50 μM N, 20 μM P and 50 μM Si — high Siconcentrations), NPSi with low Si (50 μM N, 20 μM P and12.5 μM Si— low Si concentrations). N and P additions were 3times higher than average natural concentrations of dissolvedinorganic nitrogen and total phosphorus. For silicon addition weconsidered historical data (Degobbis, 1983). Nutrient concen-trations were high enough to provide differentiation between theeffect of the added and in situ nutrients, and to generate rapidand significant response. Each microplate well was filled with200 μl of tested water samples with treatments, exposed forthree hours to UV light for sterilization, inoculated with 50 μl ofalgal inoculum solution and exposed to cultivation conditions ina Plant Growth Chamber (Voetsch, Germany).

The growth of P. tricornutum was determined by measuringthe optical density (OD) at 750 nm every day, until the stationaryphase of growth, using an automated microplate reader(Multiskan MS, Labsystem, Finland) controlled by GENESIS IIsoftware (Windows™ Based Microplate Software). The averagedaily specific growth rate (μ) of P. tricornutum, during theexponential phase of growth, which lasted up to the 7th day wascalculated from the absorbance increase per day assuming thelogarithmic increase in biomass according to ISO 10253 (1995):

A ¼lnAn=A0ð Þ= tn � t0ð Þ ð3Þwhere A0 and An were absorbencies at 750 nm at the start of thetest (t0) and after n days (tn).

The difference in the growth rate of P. tricornutum betweenenriched (μE) and control water samples (μC) is expressed as thedegree of algal response to nutrient enrichment (Δμ) and iscalculated according to Downing et al. (1999):

DA ¼ AE � AC: ð4Þ

2.5. Data analysis

Relationships among physical and chemical parameters weretested by correlation analysis. Regression analysis was per-formed for TRIX as dependent variable, with Chl-a, aD%O,DIN and TP as independent variables. Differences betweenstations based on physical and chemical parameters wereanalyzed by one-way ANOVA and Tukey's HSD test. To assessquantitatively possible nutrient limitation at each location,separate one-way ANOVAs and Tukey's HSD test wereconducted to test for significant differences in the growth rateof P. tricornutum among nutrient treatments. If the growth rate

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from a single nutrient addition was found to be significantlygreater than the growth rate in the control, the results were takento indicate limitation by that particular nutrient. If the growthrate of P. tricornutum from the combination of nutrienttreatments exceeded that of a single nutrient addition, colimita-tion was inferred. All results were considered significant at asignificance level of Pb0.05. All data except pH were logtransformed.

3. Results

3.1. Physical and chemical characteristics

Physical and chemical data are summarized in Table 1.Surface water samples between the two investigated stationswere significantly differentiated in ammonium concentrations(Tukey HSD; P=0.00002), dissolved inorganic nitrogen(P=0.01), orthophosphate (P=0.00002) and total phosphorus(Pb0.00002) concentrations. The water samples at Secchidepth and near the bottom differed significantly only inorthophosphate concentrations (P=0.00002). The surfacewater samples in Rijeka Port and Opatija contained significantlyhigher (Tukey HSD, Pb0.05) nitrate, orthophosphate and totalphosphorus concentrations, as well as significantly lower(Tukey HSD, Pb0.05) salinity, conductivity and pH in contrastto the Secchi depth and near the bottom water samples at both

stations (Table 1). Significantly higher ammonium concentra-tions (14.53±14.30 μM) were determined in Rijeka Port surfacewater in comparison to the Secchi depth (0.53±0.38 μM) andnear the bottom water samples (0.65±0.54 μM).

Chlorophyll-a concentrations varied from 0.06 in April to4.53 μg l−1 in June (mean 0.93±1.30) in the surface water, from0.29 in November to 1.98 μg l−1 in June (mean 0.90±0.64) inthe water at Secchi visibility depth and from 0.16 in Septemberto 3.7 μg l−1 in July (mean 1.07±1.10) in near the bottom waterin Rijeka Port. In Opatija, Chl-a concentrations varied from 0.12in April to 0.95 μg l−1 in June (mean 0.34±0.24) in the surfacewater, from 0.25 in July to 0.86 μg l−1 in October (mean 0.45±0.20) in the water at Secchi visibility depth and from 0.20 inSeptember to 0.70 μg l−1 in May (mean 0.38±0.17) in near thebottom water. Significant correlations were evident amongphytoplankton Chl-a and physical and chemical parameters inRijeka Port. In the surface, Chl-a was positively correlated withtemperature (r=0.82, Pb0.05) and salinity (r=0.71, Pb0.05),while in the Secchi depth and near the bottom water depth Chl-awas positively correlated with total phosphorus (r=0.80 and0.72, Pb0.05).

The surface TRIX values in Rijeka Port (from 5.3 to 6.9,mean 5.9) were significantly higher (Tukey HSD: df=16,Pb0.001) than the surface TRIX values in Opatija (from 3.1 to4.8, mean 4.1), Fig. 2. In Fig. 3 efficiency coefficients areplotted vs. trophic index values. Overall, efficiency coefficients

Table 1Average, minimum and maximum values of physical and chemical parameters in Rijeka Bay, from February 2005 to January 2006

Rijeka Port Opatija station

Surface Secchi depth Bottom Surface Secchi depth Bottom

Sec (m) 6.80 9.64(5–10) (7–14)

WD (m) 13.64 18.27(10.00–25.00) (9.00–30.00)

T (°C) 12.78a 14.56a 14.13a 13.90a 14.97a 14.75a

(7.50–20.00) (7.00–22.00) (6.50–20.00) (7.50–23.80) (8.00–22.50) (8.00–22.50)pH 7.76a 8.09b 8.05b 7.96a 8.09b 8.08b

(6.95–8.50) (7.85–8.70) (7.86–8.15) (7.85–8.13) (7.94–8.19) (7.90–8.20)EC (mS cm−1) 30.34a 51.07b 51.84b 36.26a 52.42b 52.73b

(14.14–43.60) (44.40–55.80) (46.40–56.00) (27.30–45.90) (49.80–56.10) (50.10–56.00)S 22.39a 36.78b 37.33b 24.72a 37.12b 37.20b

(8.65–36.86) (33.83–38.39) (34.30–38.39) (18.02–33.88) (35.20–38.81) (35.17–38.36)NH4 (μM) 14.53a 0.53b 0.65b 0.37b 0.27b 0.24b

(3.69–54.99) (0.00–1.25) (0.00–2.06) (0.00–0.74) (0.00–0.90) (0.00–0.53)NO2 (μM) 0.32a 0.15a 0.15a 0.14a 0.14a 0.15a

(0.13–0.82) (0.03–0.55) (0.00–0.56) (0.04–0.44) (0.02–0.49) (0.03–0.58)NO3 (μM) 37.66a 3.51b 2.53b 16.50c 2.20b 1.70b

(14.93–61.13) (1.06–7.62) (0.83–6.04) (7.19–33.16) (0.91–3.64) (0.75–3.17)o-PO4 (μM) 2.19a 0.25b 0.75b 0.19c 0.06d 0.05d

(0.32–4.08) (0.03–1.24) (0.06–4.08) (0.07–0.61) (0.00–0.29) (0.00–0.32)TP (μM) 2.52a 0.27b 0.32b 0.25b 0.15c 0.12c

(1.06–5.45) (0.11–0.57) (0.17–0.59) (0.20–0.41) (0.10–0.29) (0.09–0.16)DIN/TP 23.48a 16.26a 11.54b 59.98c 18.38a 16.49a

(11.03–44.47) (9.03–28.36) (7.05–20.04) (38.67–82.00) (6.45–21.65) (10.75–24.37)DO (mg l−1) 7.20a 6.67a 6.10a 7.36a 6.73a 7.06a

(4.75–9.96) (3.96–9.33) (3.31–8.88) (4.97–11.32) (3.76–9.46) (4.53–11.45)BOD (μg l−1) 2.81a 2.29a 2.37a 2.20a 1.90a 2.52a

(1.49–4.59) (0.25–5.57) (0.17–5.15) (0.01–4.02) (0.13–4.74) (0.34–6.29)

Different letters indicate a statistically significant difference among different water layers at two stations calculated by ANOVA and Tukey HSD at the significancelevel of Pb0.05.

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were negative, ranging from −4.8, in Rijeka Port surface, inJanuary 2006, to −0.7 near the bottom in Opatija in July 2005.Regression analysis for TRIX (dependent variables) with Chl-a,aD%O, DIN and TP (independent variables) showed that thecoefficient of determination (R2) was 0.213 for Chl-a(Pb0.001), 0.246 for aD%O (Pb0.001), 0.629 for DIN(Pb0.0001) and 0.792 for TP (Pb0.0001). Standardized partialregression coefficients (β) were significant (Chl-a: β=0.461,SE=0.124, P=0.0005; aD%O: β=0.496, SE=0.122; DIN:

β=0.794, SE=0.085, Pb0.0001; TP: β=0.89, SE=0.064,Pb0.0001).

3.2. Bioassay experiment

The growth of P. tricornutum was estimated as the averagedaily specific growth rate during the exponential phase of growth,

Fig. 2. Variations of trophic index (TRIX) and related DIN (μM), TP (μM), DO (mg L−1) and Chl-a (μg L−1) fromMay 2005 to January 2006, in Rijeka Port (a) and inOpatija (b).

Fig. 3. Efficiency coefficient vs. trophic index in Rijeka Port surface (R-surface),Secchi visibility depth (R-Secchi depth) and near the bottom layer (R-bottom);and in Opatija at surface (O-surface), Secchi depth (O-Secchi depth) and near thebottom water layer (O-bottom).

Fig. 4. The average specific growth rate (μ) of P. tricornutum (day−1) in thecontrol water samples in the Rijeka Port (a) and Opatija (b) during seven days ofbioassay experiment. Error bars represent standard deviation of six replicates.

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which lasted up to the 7th day. In surface water control samples inRijeka Port, the growth rate ofP. tricornutum varied from0.070 inJuly to 0.216 day−1 in April; in control water samples at Secchidepth from 0.052 in November to 0.271 day−1 in March, and innear the bottom control water samples from0.055 inNovember to0.283 day−1 inMarch (Fig. 4a). The growth rate ofP. tricornutumin Opatija varied from 0.060 in January to 0.274 day−1 in May inthe surface water control samples; from 0.050 in January to0.258 day−1 in May in control water samples at Secchi depth andfrom 0.041 in December to 0.256 day−1 in April in near thebottom control water samples (Fig. 4b).

After addition of N into the surface water samples from RijekaPort no significant differences were determined in the growth rateof P. tricornutum, in comparison with the control, with theexception of February and May (Fig. 5a). On the other hand,addition of P and Si alone to the same water samples caused asignificant increase in the P. tricornutum growth rate from June toDecember for P and from June to January for Si. The addition ofcombinations: NP, NPSi with low Si concentration and NPSi with

high Si concentration, caused a significant increase in theP. tricornutum growth rate in all surface water samples fromRijeka Port (Fig. 5a). InOpatija, the addition of N, P and Si singly,as well as in combinations, into the surface water samples causeda significant increase in the P. tricornutum growth rate from Juneto January, while from February toMay a significant increase wasdetermined only after the addition of NP combination (Fig. 6a). Asignificant increase in the P. tricornutum growth rate wasdetermined in Secchi depth and near the bottom water samplesfrom both stations after the addition of N, P and Si singly, and incombinations from June to January. On the other hand, fromFebruary to May only the addition of NP combinationsignificantly increased P. tricornutum growth rate (Figs. 5b andc and 6b and c).

Maximal degree of nutrient limitation at Rijeka Port wasdetermined for NP limitation (Δμ=0.240 day−1,) in Secchidepth water samples in November, Fig. 5. In Opatija, NPlimitation was also maximal (Δμ=0.249 day−1) in Secchi depthwater samples, but in January 2006, Fig. 6. Summarized results

Fig. 5. Algal response to nutrient treatments (N, P and Si, singly and in combinations) expressed as the degree of nutrient limitation (Δμ, day−1) during 7 days ofincubation. Data correspond to the surface (a), Secchi depth (b) and near the bottom samples (c) in Rijeka Port.

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of the magnitude of response to nutrient enrichment arepresented in Fig. 7. The highest average magnitude ofresponses, during the 12 months of investigation, was to theaddition of NP combination in all water samples with theexception of surface water in Rijeka Port, where a significantlyhigher magnitude of response was to the addition of NPSi withhigh Si combination.

4. Discussion

Complex and variable hydrographic and hydrodynamiccharacteristics in Rijeka Bay depend on the seasonal meteor-ological conditions, mostly inflow of freshwater (Degobbiset al., 1978; Degobbis, 1983). According to our results thedifferences between surface waters and the water column insalinity and nutrient concentrations, especially ammonium andnitrate concentrations are related to the freshwater inflow.Variability of salinity is greater in surface waters in Rijeka Port,since this sampling station was located approximately 50 m

from the coast line and in the vicinity of a sewage outfall. TheOpatija station was, on the other hand, located further offshore.Salinity is a conservative tracer, unbiased by seasonal primaryproduction and related to the nutrient content (Pirrone et al.,2005). The significantly higher ammonium concentrations thatwere determined (mostly during the colder months of the year)in the surface water at Rijeka Port, could be a result of thesewage contribution and also due to the suppression ofnitrification by low temperatures (Magnien et al., 1992).Some influence of freshwater discharge was also evident inOpatija, because of the difference in salinity and nitrateconcentrations between surface water and other depths, andalso because of the close proximity to the coast.

The maximum values of phytoplankton Chl-a concentrationsin the surface water at Rijeka Port coincided with highconcentrations of both dissolved inorganic nitrogen and totalphosphorus (Fig. 2a) as well as with maximal salinity andtemperature. This was also indicated by significant positivecorrelations between Chl-a and salinity (r=0.71, Pb0.05) as

Fig. 6. Algal growth response to added nutrients (N, P and Si, singly and in combination) expressed as the degree of nutrient limitation (Δμ, day−1) during 7 days ofincubation. Data correspond to the surface (a), Secchi depth (b) and near the bottom samples (c) in Opatija.

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well as between Chl-a and temperature (r=0.82, Pb0.05). InApril, minimal concentration of phytoplankton Chl-a in thesurface layer at Rijeka Port coincided with the lowest salinityand temperature, although concentrations of dissolved nutrientswere evenly high throughout the year (Table 1). This minimalconcentration of phytoplankton Chl-a was probably a result offreshwater inflow that caused a dilution of seawater. In theSecchi depth and near the bottom water, the positive correlationof phytoplankton Chl-a and total phosphorus indicated theimportance of phosphorus availability for phytoplanktondevelopment. The most physiologically appropriate index ofphosphorus availability is total phosphorus, because thisfraction incorporates both externally available dissolvedinorganic and organic phosphorus and internal reservesrepresented by particulate P (Sickman et al., 2003). Since inmarine systems the concentrations of dissolved organic P oftengreatly exceed the concentrations of inorganic P (Michaelset al., 1996), phytoplankton may utilize an organic source of Prather than using orthophosphates (Piehler et al., 2004). Valuesof phytoplankton Chl-a in Opatija were up to two times lowerand without peaks of any relevance when compared with Chl-avalues at Rijeka Port (Fig. 2b).

Trophic state was evaluated using the trophic index (TRIX)according to Vollenweider et al. (1998) and Giovanardi andVollenweider (2004). TRIX values range from 0 to 10 coveringa wide range of trophic conditions. Values exceeding 6 TRIXunits are typical of highly productive coastal waters; values

lower than 4 TRIX units are associated with scarcely productivecoastal water, while values lower than 3 are usually found in theopen sea (Giovanardi and Vollenweider, 2004). According tothis classification, the trophic state varied from moderate (lowerproductivity) in the coastal waters near Opatija to poor (highlyproductive) in the coastal waters of Rijeka Port (Fig. 2). Incontrolling the actual variation in TRIX data at both stations, itappears that potential productivity is dominated by nutrientcomponents, since the results suggest that 79.2% and 62.9% ofthe variability in TRIX would be associated with variation in TPand DIN, respectively. Therefore, the efficiency concept can beused to give a better interpretation of the trophic state byapplying a supplementary index in terms of a “measure of thedegree of nutrient utilization” (Giovanardi and Vollenweider,2004). As shown in Fig. 3, a distinctive cluster of points couldbe identified: Secchi depth and near the bottom water samples atboth stations (with lower trophic indices and higher efficiencycoefficients), surface waters in Opatija and surface waters atRijeka Port (with high trophic indices and low efficiencycoefficients). Thus, due to factors other than nutrients thatcontrol productivity, such as zooplankton grazing (Pickneyet al., 2001), light and temperature (Cloern, 1999), nutrients areprobably not utilized to their maximum potential.

Nitrogen is most often the principal limiting nutrient intemperate coastal marine systems (Downing et al., 1999; Herbert,1999). On the other hand, Downing et al. (1999) found thatphosphorus limitation was high in pristine, unpolluted marine

Fig. 7. Average values of the degree of nutrient limitation (Δμ, day−1) during 12 months of investigation (from February 2005 to January 2006) at two stations inRijeka Bay. Error bars represent standard deviation of the mean.

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environments as well as in those polluted with nutrients but withhigh N:P ratios. Analyses of dissolved inorganic nutrients andbioassay studies revealed phosphorus limitation in the Mediter-ranean Sea (Krom et al., 1991, 1992; Maestrini et al., 1997), aswell as in the Adriatic Sea (Pojed and Kveder, 1977; Degobbis,1990; Ogrinc and Faganeli, 2006).

In order to predict which nutrient has the potential forlimitation of phytoplankton growth, we have used the DIN/TPratio (Table 1). Values of this ratio suggest that phytoplanktongrowth in Secchi depth water samples and near the bottom watersamples at both stations should not be limited by either nutrient. Inthe surface layer in Opatija, the DIN/TP ratio indicated strong Plimitation, while in the surface layer at Rijeka Port, the DIN/TPratio suggested probable P limitation (Table 1).

Qualitative and quantitative estimation of potential N, P and Silimitation was performed using nutrient enrichment bioassay withthe unicellular diatom P. tricornutum. Silicate limitation wasconsidered due to its importance in the control of diatom growth,but it is assumed that silicate input is not significantly influencedby human activity. Based on the obtained results of the growth rateof P. tricornutum in control water samples, a separation of thesurface layer water samples from Rijeka Port coincided with asimilar separation of this station that was identified according tothe ambient nutrient concentrations and salinity as well as thetrophic conditions. Enhanced growth rate of P. tricornutum incontrol water samples at both stations from February to May incontrast to the remaining investigation periodwas probably causedby the better availability of nutrients or the efficiency of nutrientutilisation indicating greater potential for phytoplankton develop-ment in this period (Figs. 3 and 4). This means that nutrient contentsets the theoretical maximum for phytoplankton biomass, althoughthis levelmaynever be reached. In samples from these fourmonthsno limitation by either of added nutrients was determined (Figs. 5and 6). Exceptions were the surface waters at Rijeka Port wherelow colimitation of all nutrients probably caused a somewhat lowergrowth rate of P. tricornutum in the control during these fourmonths (February–May). According to Gilmartin et al. (1990)phytoplankton bloom develops in late winter and early springprimarily on nutrient reserves accumulated since late fall and earlywinter. However, there is no close relationship between the winternutrient concentration and the concentration of chlorophyll in thewater column in the summer because the dynamics of these twoparameters are completely different.

Additions of nitrogen, phosphorus and silicate alone elicitedsimilarly large average magnitudes of response in theP. tricornutum growth rate in all water samples at both stationswith the exception of the surface layer at Rijeka Port where thedegree of N limitation was not significant (Fig. 7). Thus, theresults obtained in the bioassay experiment showed that surfacewaters at Rijeka Port were rich in nitrogen content. When Ncontent is high, P becomes limiting for phytoplankton growth.According to Ault et al. (2000) silicate can have a major effecton the structure of phytoplankton communities particularly inurban coastal regions that are affected by anthropogenicdischarges rich in nitrogen and phosphorus. The growth ofmarine diatoms is limited by silicate when N:SiN1 (Justić et al.,1995; Gobler et al., 2006). There was a significantly higher

degree of NPSi (with high Si) colimitation in the surface layerwater samples at Rijeka Port. When the Si concentration wasdecreased in the NPSi (with low Si) combination, the degree ofcolimitation also decreased, indicating that Si was the primarylimiting nutrient in the surface waters at Rijeka Port. Therefore,the vicinity of sewage outfall in Rijeka Port and the high inflowof wastewaters, which are rich in nitrogen and phosphoruscontent relative to silicate, may have led to the artificial Silimitation that was also confirmed by our bioassay experiment.In contrast to the surface layer, in Secchi depth and near thebottom water samples as well as in all depths in Opatija, thehighest degree of limitation was determined when both NP andNPSi combinations were added. By decreasing the Siconcentration, the degree of the NPSi colimitation increased.Thereby, N and P limitation was equally important, while Si wasa secondarily limiting nutrient for the growth of P. tricornutum.

The northern Adriatic Sea is characterized, in general, byphosphorus limitation and by the strong development of diatompopulations, with a minor contribution by flagellates (Zavatar-elli et al., 1998). Therefore, the strong phosphorus and silicateuptake by phytoplankton and the sinking of particulate mattermay contribute to the export of the phosphorus and the silicontoward the deeper part of the water column, leaving unutilizednitrate in the upper part of the water column (Zavatarelli et al.,2000). According to Degobbis (1990), northern Adriaticphytoplankton has the ability to utilize phosphorus faster, incomparison with nitrogen and silicate, indicating that phos-phorus is the key limiting nutrient.

Because ambient nutrient concentrations are measures of theresidual nutrients after biological activity, they may not reflectactual phytoplankton nutrient limitation (Piehler et al., 2004).Measurement of nutrient concentration in the surrounding watercannot be considered the sole basis for the assessment of nutrientlimitation responsible for phytoplankton primary production dueto fact that nutrient storage in algae may occur as result of luxuryuptake (Dodds and Priscu, 1990). A basic function of enrichmentbioassay is to determine how the algae in a given test water wouldrespond to increases in certain nutrients, thereby indicating whichnutrients might be considered most likely to limit growth(Fitzgerald, 1972). However, there are a few methodologicaldrawbacks of nutrient enrichment experiments. Bioassays areperformed over a number of days under controlled conditions, andfactors other than nutrient limitation may be minimised ormagnified (Holland et al., 2004). Other factors such astemperature, light and grazing need to be considered (Elser andKimmel, 1986; Beardall et al., 2001). Also, they are mostly singlespecies experiments and thus, outcomes of this enrichmentexperiment should be extrapolated with caution to naturalecosystems since different phytoplankton communities havedifferent nutrient requirements (Howarth, 1988; Elser et al.,1990). Nevertheless, nutrient addition bioassays indicate onlylimitation under the predefined conditions.

5. Conclusion

The surface waters in Rijeka Port are characterized by the largeanthropogenic input of N and P. The TRIX values indicated Rijeka

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Port a productive coastal area as opposed to Opatija wheresignificantly lower productivity was established. The high DIN/TPratio in the surface waters indicated P limitation at both stations. Inthe Secchi depth and near the bottom samples, DIN/TP ratiossuggested no limitation of phytoplankton growth by nutrients. Inthe nutrient enrichment bioassay, P primarily limited the growth ofP. tricornutum, while N and Si rarely limited the growth.

Acknowledgements

Funding for this study was provided by the Teaching Instituteof Public Health of the Primorsko Goranska County. The Centerfor Marine Research in Rovinj, the Ruđer Bošković Institute, andthe Department of Biology in Osijek provided the infrastructurefor laboratorywork. Also, we thankGrahamMcMaster for help inlanguage editing of the manuscript. This research was (partly)supported by the Ministry of Science of Croatia. [SS]

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