Microbiological study of the Special Nature Reserve “Gornje Podunavlje” (the Upper Danube Basin) - Monoštorski rit (Monostor marsh)

Jelica Simeunović, Alpar Barši, Jelena Barbir, Petar Knežević, Olga Petrović1

Keywords: Danube, water quality, bacteria, chlorophyll-a

Introduction Upper Danube basin is a large frequently flooded valley in the north-west of Vojvodina. This area is the most important wetland in the upper course of the river Danube through Serbia. Since 2001 the Upper Danube basin has been recognized as a Special nature reserve– protected area of the 1st category. It covers an area of approximatelly 20,000 ha2 along the left bank of the Danube from the border to the village of Bogojevo (Figure 1). It consists of two large marshes, Monostor and Apatin marshes, which make an integral part with “Kopački rit” Nature Park in Croatia and National Park “Danube- Drava” in Hungary. Wetland habitats are predominant in the area with typical swamp, forest and wet meadow vegetations. The Danube and DTD Canal flow along the boundaries of the reserve, whereas the inner part of the reserve is rich in stagnant and slow-flowing waters. With its dynamics depending on the water level of the Danube, a specific swamp character of the reserve developed as a result (Stojanovic, 2002). Furthermore, a proposal for inclusion in the Ramsar list is being considered as well as for “Potential biosphere reserve network” projects. The study of the nature reserve Upper Danube basin included one-year monitoring with microbiological and Figure 1. Special Nature Reserve hydrobiological determination of quality water with the Upper Danube Basin aim of assessing ecological status of the water flooding the Monostor marsh.

Material and research methods Water sampling was performed by standard methods, seasonally, in the period from summer 2004 to summer 2005 at 6 locations: Danube - upstream from Bezdan, Danube - weekend residential area Vagoni just after the mouth of the Monostor Dunavac (Little Danube), DTD Canal - upstream from B. Monostor, DTD Canal - downstream from B. Monostor, DTD Canal – just after the Kupusinska ustava (Kupusina dyke) and ’Pcela’ – joined to the DTD Canal by Kupusina Dunavac (Little Danube). Concerning the microbiological water analysis, the heterotrophic plate count (HPC) was determined by means of cultivation method on solid Nutrient agar (Torlak, Serbia), containing peptone-1 (15 g/l), beef extract (3 g/l), sodium chloride (5 g/l), potassium phosphate (0.3 g/l) and agar (18 g/l). Incubation period for this group of bacteria was 5-7 days at a temperature of

1 Departmant of Biology and Ecology, Faculty of Natural Science, Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia

343 22°C. The HPC on the Nutrient agar served as a basis for water classification according to Kohl (1975). The thermotolerant coliform bacteria count was determined by cultivation method on Endo agar (Torlak, Serbia) after a 24-48-hour incubation period at a temperature of 44°C. In order to determine the metabolic characteristics of heterotrophic bacteria and dominant processes in the water, frequency of specific physiological groups of bacteria was determined in the heterotrophic population by replication method on appropriate media. Proteolytic, saccharolytic, amylolytic, lipolytic and hydrocarbon oxidizing groups of bacteria were analysed (Petrovic et al., 1998). Organic biodigradable water load was indicated by phosphatase activity index and classified according to a proposed water categorization (Matavulj, 1986). Chlorophyll-a concentration was determined by standard method (APHA – AWWA – WEF, 1995), and the trophication of the examined water ecosystems was assessed according to Felföldy (1980).

Results and discussion The HPC per mililiter in the water at these sampling sites ranged from 1.1x102 to 2.95x106, depending on the site and season of analyses (Graph 1.). On the basis of this parameter, an extreme was found at location Danube - Vagoni, where water quality in October 2004 corresponded to the III-IV class according to Kohl categorization (1975). This can be explained by the fact that the location is used as a weekend residential area, affected by anthropogenic activity as a result of waste disposal directly in the the Danube. At the downstream locations, the HPC decreased and the majority of sampling sites correcponded to classes II and II-III. At the last location in the sequence ('Pčela') water quality was improved.

6 ]

10 III-IV 5 III

II-III 4 )

II 3 I-II Kohl, 1975 ( 2 I 1 Class of water Heterotrophic platecaunt per ml [(log 0 Danube- Bezdan Danube- Vagoni DTD- B. DTD- B. DTD- Pc ela Monos tor Monostor Kupusinacka (upstream) (dow nstream) ustava

July, 2004. October, 2004 Mart, 2005. November, 2005.

Graph 1. HPC per mililiter in water at the sampling locations

Occurence of specific physiological groups within heterotrops is presented in Graph 2 indicating that all examined groups are present. The most dominant groups were lipid, hydrocarbon and protein degrading bacteria, whose number was greatest at location Danube -

344 Vagoni. Domination of the said physiological groups of bacteria is a consequence of specific chemical composition of the waste materials desposed into the water.

70 ] 3 60

50

40

30

20

10 Physiological group [(CFU/ml)x10 group Physiological 0 Danube- Bezdan Danube- Vagoni DTD- B. DTD- B. DTD- Pc ela Monostor Monostor Kupusinacka (upstream) (dow nstream) ustava

Saccharolytic Lypolytic (Tween 80) Proteolytic Amylolytic Hydrocarbon oxidising

Graph 2. Physiological groups of bacteria at the ampling locations

The thermotolerant coliform count (e.g. E. coli) indicated a discrepancy in water quality from sanitary and a broader ecological point of view. In most cases, the water was slightly polluted or polluted, depending on location and sampling season. At location Danube – Bezdan in October 2004 a very high level thermotolerant (fecal) coliform was registered – 5.8x105 per 100 ml and the water was very polluted from sanitary point of view (Graph 3.). There was an absence of this group of bacteria at location 'Pčela' during two seasons. Lack of correlation between this patrameter and other microbiological parameters can be explained by relativelly long viability period of coliform bacteria from the source of pollution (first two locations) to the last sampling location.

345 )

10 Highly polluted 4 Very polluted Polluted 3 per 100 ml (log 100 ml per E. coliE.

2 Moderately Weakly 1 y Water pollution (Kavka, 1994) 1994) (Kavka, pollution Water

0 Very weakl

Termotolerant coliforms- coliforms- Termotolerant Danube- Danube- Vagoni DTD- B. DTD- B. DTD- Pc ela Bezdan Monostor Monos tor Kupusinacka (upstream) (dow nstream) ustava

July, 2004. October, 2004. Mart, 2005. November, 2005.

Graph 3. Thermotolerant coliform count (e.g. E. coli)

Enzyme activity of phosphomonoesterase in water samples was used as an additional, biochemical indicator of the total organic load of water. Results of these analyses (Graph 4.) indicate that water quality at sampling locations ranged from classes II-III to IIIB according to Matavulj (1986).

7

6 )

3 5

4 IIIA IIIB

molpNP/s/dm 3

IFA ( IFA 2 Categories (Matavulj, 1986) II-III 1

0 Danube- Danube- Vagoni DTD- B. DTD- B. DTD- Pcela Bezdan Monostor Monostor Kupusinacka (upstream) (downstream) ustava

July, 2004. Mart, 2005. November, 2005

Graph 4. Phosphatase activity index at the sampling locations

Water trophication, indirectly determined on the basis of chlorophyll-a concentration, is presented in Graph 5. The values calculated indicate an accelerated eutrophication of water, resulting from antropogenic acivity at location Danube - Vagoni. Water locations downstream from Danube - Vagoni are characterized by a relatively high level of trophication (mesotrophic, meso-eutrophic and eutrophic), due to active mineralization of organic matter by heterotrophic bacteria. The highest trophication was recorded in spring, and lowest in autumn at almost all locations. The lowest trophication was found at location 'Pčela', which corresponds to the other microbiological parameters.

346

100 6 90 )

3 80 5 70 60 50 40 4 30 Chlorophyll "a" (mg/m Chlorophyll

20 1980) (Felfoldy, level Trophic 3 10 2 0 0-1 Danube- Danube- DTD- B. DTD- B. DTD- Pcela Bezdan Vagoni Monostor Monostor Kupusinacka (upstream) (downstream) ustava

July, 2004. October, 2004. Mart, 2005. November, 2005.

Graph 5. Chlorophyll-a concentration at the sampling locations Summary

On the basis of the results obtained in this study it can be concluded that the water of the Special Nature Reserve “Gornje Podunavlje” is polluted at a great extent at almost all of the sampling sites. The water quality is improved at the last location in sequence – 'Pčela', probable due to lack of pollution sources at this location and autopurification process. The results indicate that additional protection of the Special Nature Reserve “Gornje Podunavlje” should be established.

References APHA (1995): Standard methods for the examination of water and wastewater. 19th edition. Washington, 1995. FELFÖLDY, L. (1980): A biológiai vizminösités. 3. javitott és bövitte kiadás. Vizugyi Hydrobiologicalgia, 9. Budapest. MATAVULJ (1986): “The nonspecific phosphomonoester-hydrolases of microorganisms and their significance in phosphorus cycle in aquatic environments”. (In Serbian) Ph.D. Thesis, University of Zagreb, 1986. KAVKA, G.G (1994): Erfassung und Bewertung der bakteriologischen Beschaffenheit der Donau im Jahre 1993. Vergleich der Grenzprofile Deutschland-Österreich und Österreich- Slowakei. 30. Arbeitstagung der IAD, Wissenschafftliche Kurzreferate: 296.1-296.7. KOHL, W. (1975): “Über die Bedeutung bakteriologischer Untersuchungen für die Beurteilung von Fliessgewässern, dargestellt am Beispiel der österreichisch. Donau. Arch. Hydrobiol. 44(4):392-461 PETROVIĆ, O., GAJIN, S., MATAVULJ, M., RADNOVIĆ, D., SVIRČEV, Z. (1998): Microbiolosko ispitivanje kvaliteta površinskih voda. Institut za biologiju, PMF, Novi Sad. STOJANOVIĆ, V. (2002): Specijalni rezervat prirode ‘’Gornje Podunavlje’’ – geografski prikaz, zaštita i korišćenje -. Institut za geografiju, turizam i hotelijerstvo. Novi Sad.

347