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

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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
Heterotrophic plate caunt per ml [(log10]

III-IV
5
III

II-III
4
Class of water (Kohl, 1975)
II

3
I-II

2
I
1

0
Danube- Bezdan Danube- Vagoni DTD- B. DTD- B. DTD- Pcela
Monostor 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 -

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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
Physiological group [(CFU/ml)x103]

60

50

40

30

20

10

0
Danube- Bezdan Danube- Vagoni DTD- B. DTD- B. DTD- Pcela
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.

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 Termotolerant coliforms- E. coli per 100 ml (log 10)
Highly
polluted
4 Very
polluted
Polluted
3

Moderately

Water pollution (Kavka, 1994)

2

Very weakly Weakly

1

0
Danube- Danube- Vagoni DTD- B. DTD- B. DTD- Pcela
Bezdan Monostor Monostor 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).

IIIB
6

Categories (Matavulj, 1986)

5
IFA ( molpNP/s/dm3)

4
IIIA

2
II-III

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.
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 100 6

90

Trophic level (Felfoldy, 1980)

80
Chlorophyll "a" (mg/m3)

5
70
60
50
40
4
30
20
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
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Washington, 1995.
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their significance in phosphorus cycle in aquatic environments”. (In Serbian) Ph.D. Thesis,
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