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Influence of nutrients and salinity on heterotrophic and coliform bacteria in the shallow, karstic Zrmanja Estuary (eastern Adriatic

Sea)
Cilt:12 Say:46 (2003), 29-37

Jasna Hrenovic, Damir Vilicic, Bozidar Stilinovic University of Zagreb, Faculty of Science, Dept. Botany, Rooseveltov trg 6, 10000 Zagreb, Croatia,

ABSTRACT Spatial and seasonal distribution of heterotrophic bacteria (HB), proteolitic bacteria (PB) and total coliform bacteria (TC) was determined in relation to physical-chemical parameters in the karstic, highly stratified Zrmanja Estuary, eastern Adriatic Sea, in the period February to December 2000. A slight wastewater discharge resulted in increased abundance of HB, PB and TC bacteria. Abundances decreased from surface to the bottom. The autopurification was detected in the seaward 6.2 km section of the estuary, and expressed as the decrease of TIN (26%), HB (83%), PB (753%) and TC (2168%), as well as increase of water transparency (8%). Higher autopurification of water appeared during higher summer temperatures. During lower riverine water inflow, abundances of bacteria were reduced. Salinity was important environmental factor which controlled bacterial numbers. The estuary was mostly enriched with inorganic nitrogen from the Zrmanja River. Seawater was indicated as a considerable source of orthophosphates. Low concentrations of orthophosphates and high Redfield ratios indicated phosphorus as a strong limiting factor for the heterotrophic bacterial growth. Low bacterial abundances and nutrient concentrations indicated oligitrophic conditions in the upper reach of the Zrmanja Estuary. Keywords: Adriatic Sea, CFU, estuary, hererotrophic, proteolytic, total coliform bacteria.

Dou Adriyatik Denizinde Bulunan S Karstik Zrmanja Halicinde Besin ve Tuzluluk Durumlarnn Heterotrof ve Koliform Bakteriler zerine Etkisi
ZET ubat-Aralk 2000 tarihleri arasnda, Dou Adriyatik Denizinde bulunan, ileri derecede s Zrmanja Halicinde, heterotrof bakterilerin (HB), proteolitik bakterilerin (PB) ve total koliform bakterilerinin (TC) uzaysal ve mevsimsel dalmlar fiziksel ve kimyasal parametrelerle karlatrlarak tespit edilmitir. Az miktarda da olsa atk sularn bu halice boaltlmas sonucu HB, PB ve TC bakterilerinin bu sularda oalmasna neden olmutur. Bakteri younluu yzeyden derinlere doru azalmaktadr. Halicin 6,2 km lik ksmn oluturan denize doru uzanan ksmnda kendiliinden bir temizlenme tespit edilmitir. Bu blgede TIN (%26), HB (%83), PB (%753) ve TC (%2168) bakterilerinin azald ve ayn zamanda da su berraklnn (%8) artt gzlenmitir. Yksek yaz scaklarnda kendiliinden temizlenmenin artt, nehir yatanda su aknn azald dnemlerde bakteri younluunun dt tespit edilmitir. Tuzluluk bakteri saysn kontrol eden nemli bir evresel faktrdr. Hali, Zrmanja nehrinden gelen inorganik azotla zenginlemitir. Deniz suyu nemli oranda ortofosfat kayna olarak grlmektedir. Dk ortofosfat konsantrasyonu ve yksek Redfield oranlar, fosforun heterotrof bakteri bymesinde nemli bir snrlayc faktr olduunu gsterir. Dk bakteri ve besin younluu bulunmas, Zrmanja Halicinin yukar ularnn oligotrofik artlara sahip olduunu gsterir. Anahtar Kelimeler: Adriyatik denizi, CFU, hali, heterotrof, proteolitik, total Kkoliform bakterileri.

INTRODUCTION Highly stratified estuaries are maintained in areas where a high volume of river discharge is combined with low tides (Dyer, 1991). Such phenomena are well known around the Mediterranean and along the eastern Adriatic coast. There are several small karstic rivers that discharge into the eastern Adriatic Sea forming highly stratified estuaries such as Krka (Zutic and Legovic, 1987) and Zrmanja Estuary (Vilicic et al., 1999). Estuaries are highly productive habitats, providing dynamic nutrient transformation zones at the interface between freshwater and seawater (Nixon, 1995) and sediment-seawater interface (Levin et al., 2001). Heterotrophic bacteria play an important role in transformation of dissolved organic matter to biomass, and regeneration of nutrients. They represent the important link between dissolved

organic matter, particulate organic matter, larger zooplankton and bentic filter-feeders (Thingstad et al., 1999; Levin et al., 2001). Colony-forming unit (CFU) bacteria constitute a small fraction of total bacteria, but successfully indicate degradation of allochtonous and autochtonous organic matter (Puddu et al., 1998), as well as municipal or industrial pollutants (Zdanowski and Figueiras, 1997; 1999). Distribution of bacteria is affected by phisicochemical processes such as: solar radiation, water temperature, availability of nutrients (Zdanowski and Figueiras, 1997; 1999; Coelho et al., 1999), as well as biotic factors such as phytoplankton (which produce dissolved organic matter) and microheterotrophs (which graze on bacteria). Bacterial numbers are usually higher in the upper (Albright, 1983; Fuks et al., 1991) than in the middle part of estuaries (Wright and Coffin, 1983).

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Ekoloji evre Dergisi The goal of this paper is to present some spatial and seasonal variations of the abundance of heterotrophic, proteolytic and total coliform bacteria in the small karstic, highly stratified estuary. The amount of published data due to this matter has been scarce, to date. INVESTIGATED AREA The Zrmanja River is a small karstic river that discharges into the central part of the eastern Adriatic Sea. It is 69 km long, from its source in the Dinaric karstic hinterland to the mouth in the Velebit Channel. The highly stratified Zrmanja Estuary (Vilicic et al., 1999) 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. The 14 km long upper estuary (Fig. 1) extends from the Jankovica buk waterfalls to the wider portion of the middle estuary. The steep banks of the upper estuary are strongly eroded, making the estuarine bed relatively shallow (mostly about 5 m deep). The lower reach consists of the narrow strait (Novigradsko zdrilo), as a connection between the middle reach and the coastal sea (Velebit Channel). The hydrological regime of the River Zrmanja is highly variable due to rainfall variations and water losses along the open streamflow (Bonacci, 1999). The 40 years mean Zrmanja River discharge at Jankovica buk station equalls 38 m3 sec-1. Water discharge might be as high as 456 m3 sec-1 December 1959 and as low as 0.09 m3 sec-1 June 1986. There is a complex underground water circulation in the karstic Zrmanja River catchment area (Bonacci, 1999). The numerous permanent and temporary springs along the river are connected with shallow holes ("ponors") in the hinterland. Underwater springs ("vruljas") in the estuary discharge water during rainy (OctoberDecember) and snow melting periods (March-May). The tides in the area are rather weak: M2 amplitudes are below 10 cm, and K1 amplitudes are close to 13 cm (Kasumovic, 1960). The surrounding area is scarcely inhabited and

J. HRENOVIC- D. VILICIC- B. STILINOV

without considerable anthropogenic influence, resulting in moderate phytoplankton abundance (Vilicic, 1989), low chlorophyll a and nutrient concentrations as well as high content of oxygen in the estuary (Vilicic et al., 1999; 2001). MATERIAL AND METHOD Sampling was conducted at five stations (Z1, Z2, Z3, Z4, Z4A) along the upper reach of the Zrmanja River Estuary (Fig. 1) during six case studies, on February 26, April 28, Jun 21, Jul 17, October 15 and December 19, 2000. Water samples were collected using 5-liter Niskin bottles at 0.2 and 4.0 m depth. Subsamples for microbiological analyses were kept in sterile 250-ml bottles, at 4C during transport to the laboratory. Bacteriological analyses were done according to the Standard Methods for the Examination of Water and Wastewater (Anonymous, 1995). Abundances of viable aerobic heterotrophic and proteolytic bacteria were determined as colony forming units (CFU) on tryptic glucose yeast agar with addition of 15.0 g L-1 gelatine, using the spread plate method. After period of incubation (22 C/72 h), 0.46 M HgCl2 solution was added onto plates and numbers of heterotrophic and proteolitic (produced clear zones) colonies were counted. Most probable number (MPN) of total coliforms was determined by multiple-tube-inoculation procedure in the ECbroth (35C/48 h). Subsamples for phytoplankton analyses were preserved in a 2 per cent (final concentration) neutralized formaldehyde solution. The cell counts were obtained by the inverted microscope method (Utermhl, 1958). Subsamples of 50 ml were analyzed microscopically, after a sedimentation time of 24 h, within one month after the cruise. Cells were counted at a magnification of 400 x (1 to 2 transects) and 200 x (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 x. The precision of the counting method is 10%. Salinity and temperature were determined using the conductivity, temperature and depth profiler (SEA Bird Electronics Inc., USA). Nutrient concentrations were measured using standard methods (Strickland and Parsons, 1972; Ivancic and Degobbis, 1984). Total inorganic nitrogen concentration was calculated as a sum of ammonia, nitrate and nitrite concentrations. The inorganic nitrogen/ phosphorus ratio was calculated according to Redfield et al. (1963).

Figure 1. Position of stations in the upper reach of the Zrmanja River Estuary.

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Influence of nutrients and salinity on heterotrophic and coliform bacteria in the shallow, karstic ...

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Z2

The statistical analyses were done using program Statistica Version 6.0. RESULTS The bacterial abundance profiles along the upper Zrmanja Estuary are presented for each of six case studies (Figs. 2, 3, 4). Abundance of heterotrophic bacteria (HB) was low, ranging from 1 x 104 to 3.8 x 106 colonies L-1 (average of 0.46 x 106 L-1, Fig. 2). Maximum value was determined at the surface, downstream Obrovac settlement (at station Z3 in April). Remarkably increased HB abundance could be seen at other stations in April. The contribution of proteolytic bacteria (PB) in the population of HB varied between 0 and 89%, reaching maximum in April at station Z3 (Fig. 3). Abundance of total coliform bacteria (TC), as indicators of wastewaterrelated pollution, ranged from 30 to 2400 cellsL-1 (Fig. 4), with increasing values at station Z3. The numbers of HB, PB and TC were lower in summer than in other seasons and lower at 4.0 m than at the surface. The differences between bacterial numbers at the surface and 4.0 m were significant for HB and TC (p< 0.01), but not significant for PB. Frequency distribution of HB, PB and TC abundance is presented in Fig. 5. HB increased from the head of the estuary (stations Z4A and Z4) to the middle part of the upper estuary (station Z3, downstream Obrovac) and than decreased in the seaward direction (stations Z2 and Z1). Relative contribution of PB in the population of HB was relatively low at the head of the estuary and remarkably increased downstream Obrovac settlement (station Z3). After Z3 station, PB linearly decreased in the seaward direction. Frequency of TC was low around the head of the estuary, than increased at station Z3, after that decreased in the seaward direction. Generally, the highest frequencies of HB, PB and TC were found at station Z3 and gradually decreased in the seaward direction. Seasonality of temperature, salinity and riverine water inflow is presented in Fig. 6. The lowest temperatures were measured in February (7.259.48C) and the highest in June (21.64-24.51C). In June maximum temperature was detected in the marine layer, in the head of the estuary. During April and June water temperatures were higher at the surface than at 4.0 m depth. In winter the temperatures at the surface were lower than in marine layer. Minimum surface salinity (0.10 psu) was measured in October at station Z4A. Maximum salinity (36.64 psu) was determined in marine layer in December at station Z1. The inflow of riverine water into the estuary varied from 1.2 to 24.4 m3 sec-1 . The highest riverine discharge values were found during
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Z1

0.2 m 4.0 m

0
4 10 heterotrophic bacteria L
-1

Z3

Z4

0 4 Z4A 3 Feb Apr Jun Jul Oct Dec Months 2000

0 Feb Apr Jun Jul Oct Months 2000 Dec

Figure 2. The presence of heterotrophic bacteria isolated at 0.2 and 4.0 m from February until December 2000 at stations Z1, Z2, Z3, Z4, Z4A in the Zrmanja Estuary.

100 Z1 80 60 40 20 0
100 % proteolytic bacteria 80 60 40 20 0 Z3 Z4

0.2 m 4.0 m

Z2

100 80 60 40 20 0 Feb Apr

Z4A

Feb

Apr

Jun Jul Oct Months 2000

Dec

Jun Jul Oct Months 2000

Dec

Figure 3. The presence of physiological group of proteolitic bacteria in the population of heterotrophic bacteria isolated at 0.2 and 4.0 m from February until December 2000 at stations Z1, Z2, Z3, Z4, Z4A in the Zrmanja Estuary.

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25 Z1 20 15 10 5 0 25 20 15 10 5 0
Salinity (psu)

J. HRENOVIC- D. VILICIC- B. STILINOV


25 0.2 m
Temperature ( C)

0.2 m 4.0 m

Z2

4.0 m

20

15

10

Z1 Z2 Z3 Z4 Z4A

Z3

Z4

5 40 0.2 m 30
4.0 m

10 total coliforms L

-1

20

10
Feb Apr Jun Jul Oct Dec

25 Z4A 20 15 10 5 0 Feb Apr Jun Jul Oct Months 2000 Dec

Months 2000

0 25 20
Inflow (m sec )
-1

Figure 4. The presence of total coliform bacteria isolated at 0.2 and 4.0 m from February until December 2000 at stations Z1, Z2, Z3, Z4, Z4A in the Zrmanja Estuary.

Feb

Apr

Jun Jul Oct Months 2000

Dec

15 10 5 0 Feb Apr Jun Jul Oct

Figure 6. Seasonal variations of temperature, salinity and riverine water inflow in the upper reach of the Zrmanja Estuary (February-December 2000).

14 12 10 8 6 4 2 0 0-1
12 10 Frequency 8 6 4 2 0 1-20 20-40 40-60 60-80 80-100 % proteolytic bacteria

Z1

Z2

Z3

Z4

Z4A

Dec

Months 2000

1-2 2-3 6 -1 10 heterotrophic bacteria L

3-4

14 12 10 8 6 4 2 0 1-5 5-10
2

10-15 10 total coliforms L

15-20
-1

20-25

Figure 5. Frequency distribution for heterotrophic, proteolitic and total coliform bacteria at stations Z1, Z2, Z3, Z4, Z4A in the anja Estuary in the period February-December 2000.

wet period in winter (February, December) following spring (April) and autumn (October), while the lowest were during dry period in summer (Jun, July). Concentrations of total inorganic nitrogen (TIN) ranged between 1.24 and 23.06 mol L-1 with the maximum in the surface at station Z4 in December (Fig. 7). TIN concentrations were lower during summer than in other seasons and significantly lower at 4.0 m than at the surface (p< 0.05) showing the similarity with bacterial distribution (Figs. 2, 3, 4). Concentrations of orthophosphate (PO4) were low (0.01 to 0.140 mol L-1), reaching a maximum value in the lower boundary of the halocline at station Z2 in April (Fig. 7). PO4 concentrations were higher during summer than in other seasons and significantly higher at 4.0 m than at the surface (p< 0.01). The Redfield ratio ranged from 10.6 in the marine layer in April at station Z2 to extremely high values of several thousands in the freshwater layer around the head of the estuary (stations Z4 and Z4A) in winter. The calculated mean Redfield ratio was 275. When all the data were separated into two groups based on salinity values (marine and brackish data, Table 1), mean HB, PB and TC abundances were significantly higher in the brackish than in the marine
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Influence of nutrients and salinity on heterotrophic and coliform bacteria in the shallow, karstic ...

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25 0.2 m 20
TIN ( mol L )
-1

4.0 m

Z1 Z2 Z3 Z4 Z4A

15 10 5

0 0,16

0,14 0,12
PO4 ( mol L )
-1

0.2 m

4.0 m

0,1

0,08 0,06 0,04 0,02 0 Feb Apr Jun Jul Oct Months 2000 Dec
Feb Apr Jun Jul Oct Dec Months 2000

Figure 7. Seasonal variations of total inorganic nitrogen (TIN) and orthophosphate (PO4) concentrations at 0.2 and 4.0 m in the upper reach of the Zrmanja Estuary (February-December 2000).

layer. TIN concentrations were significantly higher in brackish layer, while PO4 concentrations were significantly higher in marine layer. Average temperatures during measured period were higher inthe marine layer .
Table 1. Summary of selected parameters (mean SD) and significance of the T-test between brackish and marine layers (Stations Z1, Z2, Z3, Z4, Z4A, in the period February - December 2000, N=56).

Parameter No. of samples Salinity (psu) Temperature (C) TIN (mol L-1) PO4 (mol L-1) HB (106 L-1) PB (106 L-1) TC (102 L-1)

Brackish layer 32 5.35 (6.40) 14.75 (5.51) 12.92 (6.85) 0.06 (0.03) 0.60 (0.49) 0.16 (0.15) 6.01 (8.73)

Marine layer 24 31.02 (6.02)*** 18.17 (4.91)* 5.60 (4.84)** 0.09 (0.02)** 0.15 (0.27)*** 0.03 (0.09)** 2.62 (4.84)*

TIN= total inorganic nitrogen; PO4= orthophosphate; HB= heterotrophic bacteria; PB= proteolytic bacteria; TC= total coliforms. Significance at 0.05*, 0.01**, 0.001*** levels, respectively (Student distribution).

DISCUSSION There is scarce previous information on the dynamics of CFU bacteria in highly stratified estuaries. The HB abundance is influenced by available substrates in the estuary. The supply of the substrate is a function of physical, chemical and geomorphological characteristics of the estuary. There is a paper from Ria de Vigo, Spain, where
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contribution of CFU bacteria comprised on average 1% of total bacterioplankton with a maximum of 6.8% (Zdanowski and Figueiras 1999). The abundance of CFU bacteria in Ria de Vigo varied between 8.5 x 104 and 2.5 x 108 colonies L-1. There is much more data on abundance of bacteria determined by direct counting methods. In the middle Adriatic Krka Estuary, Fuks et al. (1991) detected abundance of bacteria of 0.1 to 2.3 x 109 cells L-1, an order of magnitude higher than in the coastal and open waters of the central Adriatic, and several times lower than in the mostly eutrophicated north-west Adriatic. In the northern Adriatic Sea, which is affected by the considerable freshwater inputs, Puddu et al. (1998) reported an average heterotrophic bacterial abundance of 6.0 x 108 cells L-1. If we consider CFU abundance in the Krka Estuary as 1% of the total count, we obtain values of 1 x 106 to 2.3 x 107 colonies L-1. That means that CFU bacteria in the Krka Estuary are probable one order of magnitude more abundant than in the Zrmanja Estuary. If compared with data from other estuaries (Zdanowski and Figueiras 1999), the abundance of CFU HB in the upper reach of the Zrmanja Estuary (1 x 104 to 3.8 x 106 L-1) may be considered low. Lower bacterial abundance in the Zrmanja Estuary than those recorded in other estuarine environments is probably the consequence of low input of both allochthonous organic matter from Zrmanja River and autochthonous organic matter production in the Zrmanja Estuary. In our study, we found the increase of HB, PB and TC abundances at station Z3 after wastewater discharge from Obrovac settlement (cca 1000 inhabitants). In estuaries HB abundance may increase due to utilization of dissolved organic matter excreted by autotrophs or those of allochthonous origin (Puddu et al., 1998). Having no measured data on the availability of allochthonous organic matter, we can just suppose that bacterial abundance is indirectly linked with the input of nutrients. Increased concentration of dissolved and particulate organic matter in the area of anthropogenic influence could be indirectly indicated by the accumulation of mixotrophic dinoflagellates (70% increase) and cryptophytes (66% increase) (Fig. 8). The increased amount of dissolved organic compounds, excreted by phytoplankton could provide higher numbers of HB in definite parts of the estuary. In the Zrmanja Estuary, the maximum numbers of HB (3.80 x 106 L-1) and PB (3.23 x 106 L-1) were measured in April, probably due to the phytoplankton spring maximum (Buric et al., 2001). In the Ria de Vigo Estuary, the increased CFU values has been detected after decline
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J. HRENOVIC- D. VILICIC- B. STILINOV Table 2. Longitudinal distribution of nutrients and heterotrophic (HB), proteolytic (PB), total coliform (TC) bacteria in the upper reach of the Zrmanja Estuary.
TIN % PO4 % HB % PB % TC %

104 dinoflagellates L-1

Head (Z4A, Z4) Obrovac (Z3) Seaward portion

(mol L-1) (mol L-1) (106 L-1) (106 L-1) (102 L-1) 100 100 100 100 100 (11.80) 91 (10.78) 65 (7.66) (0.054) 144 (0.078) 156 (0.084) (0.51) 150 (0.77) 67 (0.34) (0.12) 794 (0.98) 41 (0.05) (1.20) 2493 (29.92) 325 (3.91)

104 cryptophytes L-1

(Z2, Z1)

The values at the head of the estuary represent 100 %. Absolute mean values are shown in parentheses.

Figure 8. Longitudinal distribution of phytoplankton (Dinoflagella-tes and Cryptophyceae) in the upper reach of the Zrmanja Estuary (February-December 2000).

of phytoplankton bloom and resuspension of sediment (Zdanowski and Figueiras, 1999). Increased contribution of PB in the HB population could possibly indicate anthropogenic protein input and protein decomposition by PB. Increased TC numbers, as indicators of wastewater-related pollution, indicated anthropogenic influence due to the discharge of wastewater from Obrovac settlement. According to the distribution of HB, PB and TC abundances, the upper reach of Zrmanja Estuary can be divided in three parts: head of the estuary (stations Z4A and Z4), the portion downstream Obrovac settlement (stations Z3) and seaward portion of the estuary i.e. stations Z2 and Z1 (Table 2). Remarkable abundances of HB and PB and low numbers of TC around the head of the estuary could be due to the pasture around the banks of the Zrmanja River. The highest measured abundances of HB, PB and TC at station Z3 (downstream Obrovac settlement) were due to the anthropogenic influence, while the decrease of the abundances in the seaward direction was due to autopurification process and increasing influence of salinity. The autopurification can be expressed as decrease of TIN (26%), HB (83%), PB (753%) and TC (2168%), as well as the increase of water transparency (8%) in the 6.2 km section of the upper estuary. Seaward reduction of TIN concentrations was due to the nitrogen uptake by

bacteria and phytoplankton (Middelburg and Nieuwenhuize, 2000 a, b) and downward flux mediated by migratory herbivores. In addition, the resultant nitrogen concentration in the water column is modified by microbiological nitrificationdenitrification processes (Thompson et al., 2000). Increase of PO4 concentrations in the seaward direction of the estuary was most probably due to the PO4 transport from the costal sea. In the upper reach of the Zrmanja Estuary, brackish layer provided four times higher HB, five times higher PB and three times higher TC abundance than in marine layer (Table1). Two times higher bacterial abundance in the surface low-salinity layer than in the seawater layer was detected in the Krka River estuary (Fuks et al., 1991). In the Krka River estuary higher concentrations of orthophosphates and nitrates have been detected in the surface than in the seawater layer, indicating the main input of these nutrients from the river (Fuks et al., 1991). In the Zrmanja Estuary two times higher TIN concentrations in brackish and two times higher PO4 concentrations in marine layer were measured. TIN concentrations were significantly higher in brackish than in marine layer and higher around the head of the estuary and the portion downstream Obrovac settlement, than in the seaward portion of the estuary. Pasture upstream the river is probably important source of TIN in the Zrmanja River. Another source is Obrovac settlement. Significantly higher PO4 concentrations in marine than in brackish layer, as well as in the seaward direction of the estuary, indicated seawater as the source of PO4. Correlations between bacterial abundance and other variables are presented in Table 3. Correlations among HB, PB and TC, as well as among HB, PB,
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Influence of nutrients and salinity on heterotrophic and coliform bacteria in the shallow, karstic ...

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TC and TIN concentrations were significantly positive. Especially HB and PB are depended on nutrients as a source of energy supply for maintaining, growth and multiplication (Briski et al., 2000). Heterotrophic bacteria mostly account of the inorganic nitrogen uptake in estuaries and most allochthonous organic matter is extensively microbially modified before export, burial, or consumption by higher trophic levels (Middelburg and Nieuwenhuize, 2000 a, b).

which caused cell death (Stilinovic and Futac, 1985). Higher intensity of autopurification process of water could be expected during higher summer temperatures of water. This has been consistent with previous findings of Puddu et al. (1998) who reported higher bacterial activity and shorter generation times during higher temperatures of water. Reduced abundance of bacteria could be also observed as a consequence of strong sedimentation of bacteria during low freshwater inflow (Briski et al., 2000). Higher bacterial elimination as a result of Table 3. Spearman rank correlation matrix for relationships between autopurification of water downstream the variables (N=56) in the water column at Stations Z1, Z2, Z3, Z4, Z4A from February until December 2000. Zrmanja Estuary was obtained during higher water temperatures and lower Inf Temp Sal TIN PO4 HB PB TC riverine water inflow. Inf 1.00 Variations of salinity were due to the Temp -0.87*** 1.00 riverine water discharge. The higher Sal -0.32* 0.51*** 1.00 riverine inflow resulted in the decrease of TIN 0.31* -0.60*** -0.71*** 1.00 temperature and salinity. Increased PO4 -0.38** 0.51*** 0.63*** -0.57*** 1.00 riverine water inflow during winter HB 0.22 -0.27* -0.66*** 0.55*** -0.47*** 1.00 caused destratification (disappearance of PB 0.17 -0.40** -0.65*** 0.66*** -0.47*** 0.84*** 1.00 the halocline) around the head of the TC 0.02 -0.09 -0.29* 0.38** -0.14 0.46*** 0.49*** 1.00 Inf= inflow; Temp= temperature; Sal= salinity; TIN= total inorganic estuary. TIN concentrations increased and the PO4 concentrations decreased during nitrogen; PO4= orthophosphate; HB= heterotrophic bacteria; PB= proteolytic bacteria; TC= total coliforms. Significance at 0.05*, 0.01**, higher inflow. Bacterial abundance was 0.001*** levels, respectively (Student distribution). insignificantly higher during increase inflow. Increase of bacterial abundance Correlations between HB, PB, TC and PO4 during high inflow can be due to the resuspension of concentration were negative (not significant for TC). the river sediment and terrestrial runoff from the PO4 concentration is positively correlated with surroundings area (pasture, inhabitants). salinity. Increased salinity has negative influence on Resuspension stimulate the synthesis of particulate bacterial survival (Solic and Krstulovic, 1992). organic matter in the water column, providing Assumption is that negative influence of salinity on organic substrate for bacteria and stimulate release of bacteria (due to the bacterial inactivation after the bacteria attached to particles, thus increasing bacterial osmotic shock or specific toxicity of ions) disables abundance in water column (Zdanowski and bacteria to recognise orthophosphates as nutrient. Figueiras, 1999). Terrestrial runoff enhances bacterial Establishing the significant negative correlation diversity through the introduction of terrestrial between salinity and monitored bacterial groups, bacteria or those from shallow waters (Zdanowski salinity could be important environmental factor and Figueiras, 1999; Briski et al., 2000). The increase which control bacterial numbers in the Zrmanja numbers of saprophytic and proteolytic bacteria Estuary. Salinity has been already considered major positively correlate with riverine inflow. In such factor in survival and viability of allochthonous situations, especially in oligotrophic waters, microorganisms in estuaries with high fluctuations of contribution of proteolytic bacteria can account up to salinity (Solic and Krstulovic, 1992; Hyun et al., 1999; 90% of the saprophytic bacteria abundance (Stilinovic and Futac, 1985). Turbidity peaks in the water samples Mallin et al., 2000). Due to the positive correlation of temperature and are accompanied by peaks of total coliforms in the salinity, temperature provided negative correlation seawater indicating that the contamination is with TIN concentrations and positive correlation normally deposited at the marine sediment rather with PO4 concentrations. Temperature was inversely than in the water column (Coelho et al., 1999; Briski correlated with HB, PB and TC, but it was not et al., 2000). The Redfield ratio in the upper reach of the significant for TC. Higher temperatures caused higher metabolic activity of HB, which lead to the Zrmanja Estuary was mostly much higher than faster oxidation of organic matter, oxygen deficit in established 16:1 or the typical Mediterranean ratio of water and accumulation of harmful metabolites, 22 (Thingstad and Rassoulzadegan, 1995), indicating
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Ekoloji evre Dergisi phosphorus depletion in water. Low concentrations of orthophosphates and high Radfield ratios indicate phosphorus as a strong limiting factor for the heterotrophic bacterial growth. The response to the decrease in PO4 concentrations was an increase in the Redfield ratio (p< 0.001). We emphasise that the high Redfield ratio in the upper reach of the Zrmanja Estuary is due to the very low concentrations of PO4

J. HRENOVIC- D. VILICIC- B. STILINOV

and not to increased or high concentrations of TIN. According to low bacterial abundances and nutrient concentrations the upper reach of the Zrmanja Estuary can be considered as oligotrophic. This is primarily due to the pristine waters of the karstic Zrmanja River and slight anthropogenic influence around the estuary.

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Influence of nutrients and salinity on heterotrophic and coliform bacteria in the shallow, karstic ...

Ekoloji evre Dergisi

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